Enhanced Transduction of Human Hematopoietic Stem Cells by AAV6 Vectors: Implications in Gene Therapy and Genome Editing.

We have reported that of the 10 most commonly used adeno-associated virus (AAV) serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs) in vitro, as well as in vivo. More recently, polyvinyl alcohol (PVA), was reported to be a superior replacement for human serum albumin (HSA) for ex vivo expansion of HSCs. Since HSA has been shown to increase the transduction efficiency of AAV serotype vectors, we evaluated whether PVA could also enhance the transduction efficiency of AAV6 vectors in primary human HSCs. We report here that up to 12-fold enhancement in the transduction efficiency of AAV6 vectors can be achieved in primary human HSCs with PVA. We also demonstrate that the improvement in the transduction efficiency is due to PVA-mediated improved entry and intracellular trafficking of AAV6 vectors in human hematopoietic cells in vitro, as well as in murine hepatocytes in vivo. Taken together, our studies suggest that the use of PVA is an attractive strategy to further improve the efficacy of AAV6 vectors. This has important implications in the optimal use of these vectors in the potential gene therapy and genome editing for human hemoglobinopathies such as ß-thalassemia and sickle cell disease.

Human mesenchymal stem cells-derived conditioned medium inhibits hypoxia-induced death of neonatal porcine islets by inducing autophagy.

BACKGROUND: The dysfunction of islet grafts is generally attributed to hypoxia-induced damage. Mesenchymal stem cells (MSCs) are currently thought to effectively protect cells from various risk factors via regulating autophagy. In our study, we investigated if human umbilical cord-derived MSCs could ameliorate hypoxia-induced apoptosis in porcine islets by modulating autophagy, and we explored the underlying mechanisms. METHODS: Neonatal porcine islet cell clusters (NICCs) were cultured with human umbilical cord-derived MSC conditioned medium (huc-MSC-CM) and RPMI-1640 medium (control) under hypoxic conditions (1% O2 ) in vitro. NICCs were treated with 3-methyladenine (3-MA) and chloroquine (CQ) to examine the role of huc-MSC-CM in regulating autophagy. Finally, the levels of several cytokines secreted by huc-MSCs were detected by ELISAs, and the corresponding inhibitors were applied to investigate which cytokine mediates the protective effects of huc-MSC-CM. The effects of huc-MSC-CM on NICCs viability and autophagy were examined using AO/PI staining, flow cytometry analysis, transmission electron microscopy (TEM) and confocal fluorescence microscopy analysis. The insulin secretion of NICCs was tested with an insulin immunoradiometric assay kit. RESULTS: Compared to the control, the huc-MSC-CM treatment improved the viability of NICCs, inhibited apoptosis, increased autophagic activity and the levels of PI3K class III and phosphorylated Akt, while the ratio of phosphorylated mTOR/mTOR was reduced. These changes were reversed by CQ and 3-MA treatments. High concentrations of IL-6 were detected in hu-MSC-CM. Furthermore, recombinant IL-6 pre-treatment exerted similar effects as huc-MSC-CM, and these effects were reversed by a specific inhibitor of IL-6 (Sarilumab). CONCLUSIONS: Our results demonstrated that huc-MSC-CM improved islet viability and function by increasing autophagy through the PI3K/Akt/mTOR pathway under hypoxic conditions. Additionally, IL-6 plays an important role in the function of huc-MSC-CM.

Mesenchymal stem cells alleviate hypoxia-induced oxidative stress and enhance the pro-survival pathways in porcine islets.

IMPACT STATEMENT: The utilization of mesenchymal stem cells (MSCs) is a promising approach to serve as adjuvant therapy for islet transplantation. But the inability to translate promising preclinical results into sound therapeutic effects in human subjects indicates a lack of key knowledge of MSC-islet interactions that warrant further research. Hypoxia and oxidative stress are critical factors which lead to a tremendous loss of islet grafts. However, previous studies mainly focused on other aspects of MSC protection such as inducing revascularization, enhancing insulin secretion, and reducing islet apoptosis. In this study, we aim to investigate whether MSC can protect islet cells from hypoxic damage by inhibiting ROS production and the potential underlying pathways involved. We also explore the effects of MSC-derived exosomes and IL-6 on hypoxia-injured islets. Our data provide new molecular targets for developing MSC applications, and this may ultimately promote the efficiency of clinical islet transplantation.

Human mesenchymal-stem-cells-derived exosomes are important in enhancing porcine islet resistance to hypoxia.

BACKGROUND: Hypoxia-induced damage is one of the key factors associated with islet graft dysfunction. Mesenchymal stem cells (MSCs) could be used to enhance the therapeutic effect of islet transplantation due to their paracrine potential such as exosomes. In this study, we investigated whether exosomes from human umbilical cord-derived MSC-conditioned medium (hu-MSC-CM) could increase the survival and function of neonatal porcine islet cell clusters (NICCs) exposed to hypoxia. METHODS: Neonatal porcine islet cell clusters were cultured with hu-MSC-CM, with or without exosomes, and native medium RPMI-1640 (Control) under hypoxic conditions (1% O2 ). The effects of exosomes on NICCs viability and function in vitro were examined by FACS, the Loops system, and the Extracellular Flux assay, respectively. RESULTS: Compared with NICCs cultured in RPMI-1640 medium and hu-MSC-CM without exosomes, the survival ratio, viability, and function increased in NICCs cultured in hu-MSC-CM with exosomes. CONCLUSIONS: This study found that hu-MSC-CM could protect NICCs from hypoxia-induced dysfunction, and exosomes played an important role in hypoxic resistance, suggesting a potential strategy to improve islet transplantation outcomes.

High-Efficiency Transduction of Primary Human Hematopoietic Stem/Progenitor Cells by AAV6 Vectors: Strategies for Overcoming Donor-Variation and Implications in Genome Editing.

We have reported that of the 10 commonly used AAV serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem/progenitor cells (HSPCs). However, the transduction efficiency of the wild-type (WT) AAV6 vector varies greatly in HSPCs from different donors. Here we report two distinct strategies to further increase the transduction efficiency in HSPCs from donors that are transduced less efficiently with the WT AAV6 vectors. The first strategy involved modifications of the viral capsid proteins where specific surface-exposed tyrosine (Y) and threonine (T) residues were mutagenized to generate a triple-mutant (Y705 + Y731F + T492V) AAV6 vector. The second strategy involved the use of ex vivo transduction at high cell density. The combined use of these strategies resulted in transduction efficiency exceeding ~90% in HSPCs at significantly reduced vector doses. Our studies have significant implications in the optimal use of capsid-optimized AAV6 vectors in genome editing in HSPCs.

The effects of endothelial progenitor cells on rat atherosclerosis.

Atherosclerosis (AS) is a progressive disease characterized by endothelial injury and lipid aggregation in the arterial walls. Studies have reported that endothelial progenitor cells (EPCs) derived from the bone marrow (BM) might provide an endogenous repair mechanism by differentiating into endothelial cells to replace the dysfunctional endothelium. Our study aims to investigate the effect of EPCs derived from rat BM on AS. EPCs transduced by recombinant adeno-associated virus-green fluorescent protein (GFP) were transplanted into a rat AS model. After 2 months of transplantation, the localization of GFP-labeled cells, morphology, and lipid content in the aorta were examined. GFP-labeled EPCs were found in the endothelial monolayer of the artery vessel in the GFP/EPC group. Hematoxylin and eosin staining suggested that the lipid deposits in the aortic endothelium in the EPC/GFP group were less compared with those in the untreated group. Oil Red O staining of liver slices showed that lipid droplets were obviously decreased in the GFP/EPC group. The endothelial nitric oxide synthase and apolipoprotein E mRNA levels in the GFP/EPC group were significantly higher, but the intercellular cell adhesion molecule-1 mRNA level was significantly lower compared with the control group. The results suggest that EPCs derived from the BM can repair the injured endothelium and promote an atherosclerotic lesion regression. Therefore, EPCs may provide a useful tool for the treatment of AS.

Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo.

BACKGROUND AIMS: Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. METHODS: We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. RESULTS: We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo. CONCLUSIONS: These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.

Optimization of the capsid of recombinant adeno-associated virus 2 (AAV2) vectors: the final threshold?

The ubiquitin-proteasome pathway plays a critical role in the intracellular trafficking of AAV2 vectors, and phosphorylation of certain surface-exposed amino acid residues on the capsid provides the primary signal for ubiquitination. Removal of several critical tyrosine (Y) and serine (S) residues on the AAV2 capsid has been shown to significantly increase transduction efficiency compared with the wild-type (WT) vectors. In the present study, site-directed mutagenesis of each of the 17 surface-exposed threonine (T) residues was conducted, and the transduction efficiency of four of these mutants, T455V, T491V, T550V, and T659V, was observed to increase up to 4-fold in human HEK293 cells in vitro. The most critical Y, S, and T mutations were subsequently combined, and the quadruple-mutant (Y444+500+730F+T491V) AAV2 vector was identified as the most efficient. This vector increased the transduction efficiency ∼24-fold over the WT AAV2 vector, and ∼2-3-fold over the previously described triple-mutant (Y444+500+730F) vector in a murine hepatocyte cell line, H2.35, in vitro. Similar results were obtained in murine hepatocytes in vivo following tail vein injection of the Y444+500+730F+T491V scAAV2 vector, and whole-body bioluminescence imaging of C57BL/6 mice. The increase in the transduction efficiency of the Y-T quadruple-mutant over that of the Y triple-mutant correlated with an improved nuclear translocation of the vectors, which exceeded 90%. These observations suggest that further optimization of the AAV2 capsid by targeting amino acid residues involved in phosphorylation may not be possible. This study has thus led to the generation of a novel Y444+500+730F+T491V quadruple-mutant AAV2 vector with potential for use in liver-directed human gene therapy.

High-efficiency transduction of primary human hematopoietic stem cells and erythroid lineage-restricted expression by optimized AAV6 serotype vectors in vitro and in a murine xenograft model in vivo.

We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34(+) cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: ß-globin promoter (ßp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the ß-globin locus control region (HS2-ßbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-ßp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34(+) cells in vitro. Transgene expression from the B19p6 or the HS2-ßp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-ßp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-ßp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as ß-thalassemia and sickle cell disease.

Limitations of encapsidation of recombinant self-complementary adeno-associated viral genomes in different serotype capsids and their quantitation.

We previously reported that self-complementary adeno-associated virus (scAAV) type 2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids. Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained. Recently, it has been reported that quantitative real-time PCR assays may result in substantial differences in determining titers of scAAV vectors depending on the distance between the primer sets and the terminal hairpin structure in the scAAV genomes. We also observed that the vector titers determined by the standard DNA slot-blot assays were highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly overrepresented compared with the probes hybridizing close to the middle of the viral genomes. These differences among various probes were not observed using Southern blot assays. This overestimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes. Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes. Although the discrepancy is modest with wild-type serotype capsid and short viral genomes, the measured titer could be as much as fivefold different with capsid mutant vectors and large genomes. Thus, based on our data, we suggest that Southern blot analyses should be performed routinely to more accurately determine the titers of recombinant AAV vectors. At the very least, the use of probes/primers hybridizing close to the mutant inverted terminal repeat in scAAV genomes is recommended to avoid possible overestimation of vector titers.

Adeno-associated virus serotype 2 mediated transduction and coexpression of the human apoAI and SR-BI gene in HepG2 cells.

Cholesterol efflux is the first step in the reverse cholesterol transport (RCT) pathway, removing excess cholesterol from tissues, including the arterial wall, thus preventing the development of atherosclerosis. Adeno-associated virus (rAAV) has demonstrated significant promise as a DNA-delivery vector to treat serious human diseases. In this study, we constructed recombinant adeno-associated viruses coexpressing apoAI and SR-BI successfully, the double gene mRNA and protein were both strongly expressed in transduced HepG2 cells. A novel safe and efficient method of promoting the reverse cholesterol transport (RCT) may be established. These results may provide a new method for gene therapy of Arteriosclerosis.

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.

Treatment of atherosclerosis by transplantation of bone endothelial progenitor cells over-expressed paraoxonase-1 gene by recombinant adeno-associated virus in rat.

Endothelial dysfunction/loss is a key event in the development of vascular diseases, including native atherosclerosis (AS). Recent studies have shown that endothelial progenitor cells (EPCs) have the ability to repair endothelial cells that have been lost or damaged following AS. As a result, the therapy of transplanting EPCs is a promising option for the treatment of AS. However, the therapeutic effect on AS with only EPCs transplantation has not been satisfactory. The upregulation of those genes, which prevent the progress of AS in EPCs, is a novel therapeutic strategy for AS. Because it can reduce macrophage foam cell formation and protect endothelial cells from the oxidation of low-density lipoprotein (ox-LDL), paraoxonase-1 (PON1) gene is a candidate for gene therapy in AS. In this study, a recombinant adeno-associated virus (rAAV) vector carrying the human paraoxonase-1 (hPON1) gene (rAAV-PON1) was constructed, and endothelial progenitor cells (EPCs) transfected with rAAV-PON1 were transplanted into the atherosclerosis model of Sprague-Dawley rats (SD rats). The results of doppler ultrasound and histological analysis showed that the group transplanted with the hPON1 gene-transfected EPCs was superior to the group transplanted only with the EPCs and was also better than the group with hPON1 gene injection alone. The results indicated that rAAV-mediated hPON1 gene-transfected EPCs is a potentially valuable new tool in the treatment of atherosclerosis.

[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.