This study was aimed to explore the infection characteristics of murine mononuclear cell subpopulations in bone marrow with murine cytomegalovirus (MCMV). Subpopulations of mononuclear cells, including lin(+), lin(-), lin(-)CD117(+) and lin(-)CD117(-) cells, were infected with MCMV after being separated by MACS, and induced to differentiation by adding cytokines or inducer, then nucleic acid and proteins were detected. The results indicated that the MCMV DNA, IE transcripts and IE protein could be detected in the lin(+) cells infected with MCMV; no virus products were detected in infected lin(-) cells without adding any stimulating factors, while IE and E transcripts and proteins were detected after adding GM-CSF, rhEPO or phorbol ester in the lin(-) cells infected with MCMV. Furthermore, no IE or E gene transcripts were detected in the lin(-)CD117(+) and lin(-)CD117(-) cells, but the cell colony formation of lin(-)CD117(+) hematopoietic stem and progenitor cells was inhibited after MCMV infection and expression of CD117 antigen on cell surface of the lin(-) cells was downregulated. It is concluded that MCMV can latently infect subpopulations of mononuclear cells in the murine bone marrow. Cells which are of characteristics of primitive stem and progenitor cells are not susceptible to MCMV, but infection of these cells with MCMV can inhibit functions of cells and downregulate the expression of antigen on cells surface.
Animals ; Bone Marrow ; virology ; Cytomegalovirus Infections ; Mice ; Mice, Inbred BALB C ; Monocytes ; virology ; Muromegalovirus ; physiology ; Proto-Oncogene Proteins c-kit ; Stem Cells ; virology

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.
Antigens, CD34 ; metabolism ; Bone Marrow Cells ; cytology ; metabolism ; virology ; Dependovirus ; genetics ; Genetic Therapy ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Hematopoietic Stem Cells ; metabolism ; virology ; Humans ; Mesenchymal Stem Cells ; metabolism ; virology ; Recombination, Genetic ; Transduction, Genetic

OBJECTIVETo compare the transduction efficiencies of adenoviral vector, adeno-associated viral vector, baculoviral vector, and plasmid vector in human bone-marrow-derived mesenchymal stem cells (hBMSCs).

METHODSThe hBMSCs were cultured in vitro and transducted with the adenoviral vector, adeno-associated viral vector, baculoviral vector, and plasmid vector. The expression of target protein was observed by inverted fluorescent microscopy and flow cytometry.

RESULTSInverted fluorescent microscopy showed that some of the hBMSCs after transduction expressed the green fluorescent protein (GFP) and the hBMSCs transducted with baculoviral vector expressed more GFP than those of other three vectors. Flow cytometry showed that the transduction efficiencies and mean fluorescence intensities of the adenoviral vector, adeno-associated viral vector, and plasmid vector were 42%, 37%, and 22% and 158, 115, and 77, respectively, which were significantly lower than those of baculoviral vector (70%, P < 0.01; 212, P < 0.05; respectively).

CONCLUSIONCompared with the adenoviral vector, adeno-associated viral vector, and plasmid vector, the baculoviral vector has higher transduction efficiency in hBMSCs and therefore may be a more suitable gene vector for research in human gene therapy.

Adenoviridae ; genetics ; metabolism ; Baculoviridae ; genetics ; metabolism ; Bone Marrow Cells ; metabolism ; virology ; Cells, Cultured ; Dependovirus ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Hematopoietic Stem Cells ; metabolism ; virology ; Humans ; Plasmids ; genetics ; metabolism ; Transduction, Genetic ; methods

OBJECTIVETo investigate the efficient transfection of green fluorescent protein gene (GFP) mediated by recombinant adenovirus vector(Ad-GFP) to rat bone marrow mesenchymal stem cells (MSCs) in vitro.

METHODSWistar rat bone marrow-derived MSCs were separated and purified in vitro by Percoll density gradient centrifugation combined with adherent cell culture followed by identification with flow cytometry. MSCs infected by Ad-GFP were observed and the transfection efficiency was assessed by fluorescence microscope. The proliferative ability of these cells was tested by CCK-8.

RESULTSThe transfection efficiency was as high as 90.0%. Expression of GFP gene of infected MSCs was stable for 1 month after infection. There was no statistically difference in proliferative ability between the infected MSCs and non-infected ones (P>0.05).

CONCLUSIONThe infected MSCs with Ad-GFP expressed GFP with high efficiency and retain the ability of proliferation as non-infected MSCs. Transgection with Ad-GFP is a highly effective method for labeling MSCs.

Adenoviridae ; genetics ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; metabolism ; virology ; Cell Proliferation ; Cells, Cultured ; Female ; Genetic Vectors ; genetics ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Male ; Mesenchymal Stromal Cells ; cytology ; metabolism ; virology ; Mice ; Rats ; Rats, Wistar ; Transduction, Genetic ; methods

Adenoviridae ; genetics ; Animals ; Bone Marrow Purging ; methods ; Genes, p53 ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic Stem Cells ; cytology ; virology ; Humans ; Receptors, Virus ; genetics ; Transplantation, Autologous ; methods

OBJECTIVETo evaluate the therapeutic effect of in vitro induced autologous bone marrow-derived liver stem cell transplantation for posthepatitic cirrhosis.

METHODSBetween Jun 2008 and Mar 2009, 12 patients with posthepatitic cirrhosis and portal hypertensive underwent azygousportal disconnection and splenectomy in our department. The patients were then divided into two groups to receive autologous bone marrow-deprived liver stem cell infusion via the hepatic artery after in vitro induction for 7 days (n=6) or saline (n=6). The therapeutic effects of the operations on the liver functions and liver fibrosis index were evaluated.

RESULTSAll the patients recovered uneventfully and no side effect of the operation was found. After the operation, the patients receiving bone marrow-deprived liver stem cell infusion showed better hepatic function improvement than those receiving saline infusion (P<0.05).

CONCLUSIONTransplantation of in vitro induced autologous bone marrow-derived liver stem cell via the hepatic artery is safe and effective for treatment of posthepatitic cirrhosis.

Adult ; Bone Marrow Cells ; cytology ; Female ; Hepatitis, Viral, Human ; complications ; Humans ; Liver Cirrhosis ; etiology ; therapy ; virology ; Male ; Middle Aged ; Stem Cell Transplantation ; Transplantation, Autologous

To culture bone marrow mesenchymal stem cells (BMSCs) of rat in vitro and observe HSV-1 infection on BMSCs, BMSCs were separated from the bone marrow and identified by alizarin red staining and detection of ALP. The morphology of HSV-1 infected BMSCs and the CPE were observed. The total DNA was extracted from HSV-1 infected BMSCs and the desired specific gene fragment of 477bp of HSV-1 was amplified by PCR. Results showed that after BMSCs were induced by mineral-fluid for 14 days, the ALP level was increased and the nodule calcification was formed. The induced BMSCs were manifested to have the characteristics of osteoblasts. CPE couldn't be found in HSV-1 latently infected BMSCs but the 477bp gene fragment was still detectable. HSV-1 could establish latent infection in BMSCs after 7 passages. This study indicated that rat BMSCs could be induced to differentiate into osteoblasts in vitro, therefore they can be used as the seed cells for the tissue engineering. HSV-1 can infect rat BMSCs and develop the latent infection in vitro.
Alkaline Phosphatase ; analysis ; Animals ; Bone Marrow Cells ; virology ; Cell Differentiation ; Female ; Herpesvirus 1, Human ; physiology ; Male ; Mesenchymal Stromal Cells ; cytology ; virology ; Polymerase Chain Reaction ; Rats ; Rats, Wistar ; Tissue Engineering ; Virus Latency

BACKGROUNDTo observe cytopathogenic effect of Hantaan virus (HV) on cultured human bone marrow cells.

METHODSLight and transmission electron microscopy and direct immunofluorescent technique were applied to study cellular structure especially ultrastructural changes of bone marrow cells from patients with Hantaan virus infection. Bone marrow cells of one healthy volunteer were also studied as control.

RESULTSThe antigen of HV was found in bone marrow cells of 20 of 27 HFRS patients by the aid of direct immunofluorescent technique. It was found that the granulocytes had the highest percentage of HV antigen positive cells (76%), followed by monocytes (65%), lymphocytes (40%), megakaryocytes (20%) and the lowest was found in erythrocytes (3.7%). The injury of cell membrane after infection with HV was significantly more severe than that in the control group under the light and electron microscopy.

CONCLUSIONThis study demonstrated that HV could attack human bone marrow cells and cause cytopathogenic effect on them.

Adult ; Aged ; Antigens, Viral ; analysis ; Bone Marrow Cells ; ultrastructure ; virology ; Female ; Fluorescent Antibody Technique, Direct ; Hantavirus ; immunology ; pathogenicity ; Hemorrhagic Fever with Renal Syndrome ; pathology ; virology ; Humans ; Male ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Middle Aged

Mesenchymal stem cells (MSCs) have received considerable attention for various therapeutic approaches in recent years. MSCs are also easy to genetically modify to express therapeutic genes by using lentiviral vectors. Because of the similarities in genetics, physiology and metabolism between non-human primates (NHPs) and humans, NHPs models are invaluable for researching human disorders and for developing therapeutic strategies. Therefore, MSCs derived from NHPs could be a powerful tool for cell therapy and genetic engineering. Studies from captive and free-ranging adult NHPs show that up to 100% were infected with simian foamy virus (SFV). In this study, we found that all cultured MSCs derived from adult cynomolgus monkey were infected with SFV by RT-PCR. Therefore, antiviral drugs must be added in MSCs culture. However, because of SFV infection and additive antiviral drugs, the infection efficiency of the lentiviral vectors reduced significantly. In this study, we improved the infection efficiency by disabled antiviral drugs before lentiviral infection. It might be provide technical assistance for the culture of adult cynomolgus monkey MSCs as genetically engineered cells applied to clinical and experimental research.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Lentivirus ; genetics ; Macaca fascicularis ; Mesenchymal Stromal Cells ; cytology ; virology ; Simian foamy virus ; physiology ; Transduction, Genetic

BACKGROUNDGallbladder carcinoma (GBC) has a high mortality rate, requiring synergistic anti-tumor management for effective treatment. The myxoma virus (MYXV) exhibits a modest clinical value through its oncolytic potential and narrow host tropism.

METHODSWe performed viral replication assays, cell viability assays, migration assays, and xenograft tumor models to demonstrate that bone marrow-derived stem cells (BMSCs) may enhance efficiency of intravenous MYXV delivery.

RESULTSWe examined the permissiveness of various GBC cell lines towards MYXV infection and found two supported single and multiple rounds of MYXV replication, leading to an oncolytic effect. Furthermore, we found that BMSCs exhibited tropism for GBC cells within a Matrigel migration system. BMSCs failed to affect the growth of GBC cells, in terms of tumor volume and survival time. Finally, we demonstrated in vivo that intravenous injection of MYXV-infected BMSCs significantly improves the oncolytic effect of MYXV alone, almost to the same extent as intratumoral injection of MYXV.

CONCLUSIONThis study indicates that BMSCs are a promising novel vehicle for MYXV to clinically address gallbladder tumors.

Animals ; Bone Marrow Cells ; cytology ; Cell Movement ; physiology ; Cell Survival ; physiology ; Female ; Gallbladder Neoplasms ; therapy ; virology ; Humans ; Immunohistochemistry ; Mice ; Myxoma virus ; pathogenicity ; Stem Cells ; cytology ; physiology ; Virus Replication ; physiology ; Xenograft Model Antitumor Assays