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Wen Xing, Peng-Xia Liu,
Meng Liu,
Shao-Guang Yang,
Qin-Jun Zhao,
Jian-Ping Li,
Shi-Hong Lu,
Hong-Ying Ren,
Ying Huang,
Hao Wu,
Bin Liu,
Lei Zhang,
Zhong-Chao Han
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ABSTRACT: Bone marrow (BM) is the major source of mesenchymal stem cells (MSC). In most experiments, MSC were classically cultured from mononuclear cells (MNC) isolated by density gradient centrifugation method. However, several studies have demonstrated that this method was less efficient for MSC recovery. This study was aimed to investigate whether BM particles were the cause resulting in less efficiency of this method and how to isolate them. A total of 20 patients were enrolled in this study. MNC were cultured by standard adherence and BM particles were cultivated by primary explant culture. For BM from patients 1-10, MNC were first isolated and BM particles were then filtered out. The morphology and the fibroblastic colony number were compared between cultures of MNC and BM particles. For BM from patients 11-20, MNC isolation and BM particle filtration were processed in opposite order, then the immunophenotype and function between adherent cells expanded from MNC and BM particles were compared. In addition, for patients 11-20, the left BM aspirates were cultured too after BM particles and MNC were isolated separately. The results showed that adherent cells from BM particles were MSC. After BM particles were filtered out and cultured separately, MSC could be recovered completely from MNC isolated by density gradient centrifugation and no MSC were left in the residual BM aspirates. BM particles, which have been mostly discarded by the method of density gradient centrifugation, are another important source of MSC and they can be cultivated reliably by primary explant culture. It is concluded that more MSC are recovered from a single BM sample by culturing BM particles and MNC separately.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 11/2010; 18(6):1552-9.
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ABSTRACT: This study was aimed to construct an adenovirus hybrid system with high transduction efficiency and site-specific integration. By a series of DNA manipulation, a hybrid system of two adenovirus vectors was constructed. One vector contains loxP-flanked transgene expression cassette, in which there are hFIX and DsRed coding sequences and attB for phiC31 recolonization. The other vector carries Cre and phiC31 gene. Vectors only expressing Cre or phiC31 were used as controls. 293A cells were constructed and transfected with the adenoviral vectors by Lipofectamine 2000, and the expression of target genes was identified by fluorescence microscopy and RT-PCR. The results showed that after being identified by PCR, restriction analysis and sequencing, an adeno-integrase hybrid system was successfully constructed. The system expressed RFP, GFP, hFIX, Cre and phiC31 in 293A cells in vitro. It is concluded that the adeno-integrase hybrid system is successfully constructed, which lays a good foundation for further investigation of its therapeutic application.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 10/2010; 18(5):1229-34.
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ABSTRACT: OBJECTIVE: To investigate whether the plasmid bearing attB and human coagulation factor IX (hFIX) coding sequence could insert into hemophilia B mice genome and persistently express hFIX with co-injected integrase. METHODS: The plasmid attB-hFIX-pIRES2-EGFP was constructed, which bore attB site and hFIX coding sequence and was proved in vitro to express hFIX. The plasmid and CMV-int expressing integrase was co-infused rapidly in a large-volume solution through tail vein of hemophilia B mice. Mice infused with the plasmid alone served as controls. ELISA was performed to determine serum hFIX level. Correction of coagulation defect in vivo by plasmid infusion was assessed by bleeding time. Genomic integration of the plasmid was determined by nested PCR. RESULTS: The plasmid attB-hFIX-pIRES2-EGFP was successfully constructed. The hemophilia B mice produced (1533 ± 239) ng/ml hFIX at 24 hour after infusion of the hFIX encoding plasmid and the bleeding diathesis of the hemophilia B mice was significantly corrected as measured by clotting assays. However, whether or not co-injected with CMV-int, the serum hFIX level decreased to background level in 10 days after infusion. Nested-PCR results indicated that the integrase phiC31 resulted in the integration of the plasmid in the mouse liver chromosomes. CONCLUSION: Integrase phiC31 can catalyze recombination of 34 bp attB and pseudo-attP. Human FIX driven by CMV promoter can be transiently and highly expressed after infusion, but rapidly silenced in vivo.
Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 05/2010; 31(5):294-299.
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ABSTRACT: In order to evaluate whether mesenchymal stem cells (MSCs) from non-hematopoietic tissues are able to regulate megakaryocytopoiesis, we identified human MSCs from adult bone marrow (ABM), fetal pancreas (FPan) and umbilical cord (UC), and their abilities to support megakaryocyte (MK) differentiation from CD34(+) hematopoietic progenitor cells (HPCs) were comparatively studied. First, MSCs were isolated from ABM, FPan and UC then their growth kinetics, molecular characterization and mesodermal differentiation capacity were determined. ABM-MSCs, FPan-MSCs and UC-MSCs were irradiated and cocultured with human umbilical cord blood (UCB) CD34(+) cells, and the expansion efficiency of MK progenitor cells and MK formation were analysed and compared. Finally, SCF, IL-6 and GM-CSF expression by the three types of MSCs were also examined. Our results showed that FPan-MSCs and UC-MSCs shared most of the characteristic of ABM-MSCs, including morphology, immunophenotype, adipogenic and osteogenic differentiation potentials. Compared with ABM-MSCs, fetal MSCs had higher proliferative capacity. After 7 days' coculture, the maximal production of CD34(+)/CD41a(+) cells was obtained in a group of CD34(+) HPCs + ABM-MSCs. Furthermore, this group produced more MK colonies than other groups (p < 0.05). Surface antigen and ploidy analysis morphological observation demonstrated that a proportion of expanded cells in each group differentiated into mature MKs. ABM-MSCs, FPan-MSCs and UC-MSCs were revealed to express SCF, IL-6 and GM-CSF at mRNA level. We conclude that FPan-MSCs and UC-MSCs have the ability to promote megakaryocytopoiesis, while ABM-MSCs expand more MK progenitor cells from CD34(+) HPCs than MSCs from non-hematopoietic tissues and CD34(+) cells alone.
Platelets 02/2010; 21(3):199-210. · 1.85 Impact Factor
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ABSTRACT: The present study was aimed to isolate and identify human mesenchymal stem cells from adult bone marrow (BM-MSC) and umbilical cord (UC-MSC), and to compare their ability to support in vitro long-term hematopoiesis. MSC from bone marrow and umbilical cord were isolated by using density gradient centrifugation or enzyme digestion. MSC were further purified by adherent culture. Immunophenotype, adipogenic and osteogenic differentiation potential of BM-MSC and UC-MSC were detected. The hematopoietic supporting capacity of BM-MSC and UC-MSC was assessed by LTC-IC assay. Nonadherent cells in each group were collected for phenotypic analysis at 3, 5 and 7th week of culture. The results showed that BM-MSC and UC-MSC in culture shared a similar spindle-shaped morphology and adhered to the tissue culture substrate. They were both positive for CD90, CD105, CD73, CD29, CD54, CD166, HLA-ABC, and negative for HLA-DR, CD34 and CD45. BM-MSC and UC-MSC could differentiate into adipocytes or osteoblasts confirmed by oil red O staining and von Kossa staining, separately. LTC-IC assay showed that at 5th week of culture, the difference of the CFC yields between UC-MSC group and BM-MSC group was not statistically significant (p>0.05). At 6, 7, 9th week of culture, the CFC yields in the UC-MSC group were lower than those of BM-MSC (p<0.05). The phenotypic analysis of nonadherent cells at 3, 5, 7th week of culture indicated that along with prolongation of time, the percentages of CD34+ cells and CD117+ cells in each group decreased markedly, and the percentages of CD33+ cells, CD13+ cells and CD11b+ cells increased gradually. It is concluded that MSC from human adult bone marrow and umbilical cord can be successfully isolated and identified. UC-MSC are able to support long-term hematopoiesis in vitro, but its hematopoietic supportive capacity is weaker than those of BM-MSC.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 10/2009; 17(5):1294-300.
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ABSTRACT: This study was aimed to design and screen short hairpin RNA (shRNA) molecules targeting multidrug resistance gene (mdr1), as well as to investigate the effects of shRNA expression vector on K562/A02 cells. Mdr1-shRNA expression vector was transfected into K562/A02 cells by lipofectamine 2000, and G418 was added to screen and establish the stable expression cell strain. The expressions of mdr1 mRNA and protein were detected by real-time RT-PCR and Western blot respectively. The sensitivity of cells to chemodrugs after interference were tested by CCK8 assay. The function of p-glycoprotein was determined by Rhodamine 123 efflux experiment. The results showed that all of 4 mdr1-shRNA expression vectors could significantly knockdown the expression of p-glycoprotein as compared with control vector, moreover, the vector targeting 508 - 526 sites of mdr1 gene was the best one. It is concluded that the mdr1-shRNA expression vector gained by screening can significantly knockdown the expression of mdr1 gene and reverse leukemia drug resistance, paving the way for the application of RNAi in the following animal experiments.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 07/2009; 17(3):563-7.
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ABSTRACT: The present study was purposed to evaluate the safety of mesenchymal stem cell (MSC)-based therapy impacting on atherosclerosis. Allogeneic MSCs were obtained from rabbit bone marrow aspirates and expanded in vitro. New Zealand white rabbits were divided into three groups: 24 rabbits with hypercholesterolemia receiving intravenous injection of either 5 x 10(7) MSCs (n = 12) or saline (n = 12) after 5 weeks on a high lipid diet and additional rabbits (n = 6) fed with standard rabbit diet were served as controls. Body weight and blood lipids were measured at weeks 0, 5, 9 and 13 during the study. All rabbits were sacrificed at week 13. Atherosclerotic lesion size and vasa vasorum were evaluated by using pathological analysis and immunocytochemical technique. The results showed that the aortic sinus lesion size significantly increased in rabbits infused with MSCs as compared with controls receiving saline (23.35 +/- 3.51% and 11.39 +/- 3.08% respectively). The lesion size in whole aortas of MSC-treated rabbits was 76.64 +/- 12.70% versus 57.61 +/- 9.00% in saline-treated animals (p < 0.05). Moreover, vasa vasorum networks in MSC-treated aortas were more numerous and had increased capillary density. It is concluded that the allogeneic MSC transfusion may result in an increase in atherosclerotic lesion size. In cell therapy with MSCs or cell populations containing MSCs a strategy to attenuate the high potential of MSCs involved in atherogenesis of atherosclerosis should be taken in account.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 06/2009; 17(3):700-5.
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ABSTRACT: We have recently provided evidence that transplantation of G-CSF mobilized peripheral blood mononuclear cells (M-PBMNCs) improves limb ischemia in diabetic patients. This method represents a simple, safe, effective, and novel therapeutic approach for diabetic ischemia. Here we investigated the mechanisms by which mobilized blood cells transplantation improves limb ischemia. Unilateral hindlimb ischemia was surgically induced in streptozotocin-induced diabetic nude mice, and they were intramuscularly injected 10(6) M-PBMNCs, or human umbilical vein endothelial cells (HUVECs), PBS controls. We compared their blood-flow restoration via laser Doppler perfusion image (LDPI), angiogenesis via histological determination of capillary density. Physiological and histological assessment revealed an acceleration of ischemia recovery and increase in capillary density with less apoptosis in M-PBMNCs group, compared with those in HUVECs and PBS groups. In vivo noninvasive imaging and immunofluorescence revealed the survival, migration, proliferation, differentiation, and incorporation of M-PBMNCs into foci of vessel networks. More angioblasts were from blood cells after mobilization, and they also produced a number of antiapoptotic and proagniogenic factors that promoted angiogenesis in vivo. M-PBMNCs and its conditioned medium augmented the vessel formation in matrigel plugs in vivo. Thus, transplantation of M-PBMNCs achieved therapeutic neovascularization via supply of abundant angioblasts and angiogenic factors.
Journal of Cellular Biochemistry 09/2007; 102(1):183-95. · 2.87 Impact Factor
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ABSTRACT: Previous in vitro studies have revealed that oxidized low density lipoprotein (OxLDL) has negative effects on the proliferation and activity of endothelial progenitor cells (EPCs). Here, we evaluated the effect of OxLDL on the therapeutic potential of EPCs in ischemia-induced neovascularization. EPCs derived from mobilized human peripheral blood mononuclear cells were cultured without or with OxLDL before transplantation. Hindlimb ischemia models were surgically induced in athymic nude mice, which then received an intracardiac injection of 3 x 10(5) EPCs. By laser Doppler perfusion image and ischemia damage score, we found that blood perfusion and ischemia damage were less well recovered in the OxLDL-treated EPC transplantation group than in controls. Histological examination showed fewer transplanted EPCs and lower capillary density in ischemic tissue. Local delivery of Stromal cell-derived factor (SDF-1) restored this defect and improved blood perfusion by recruiting OxLDL-treated EPCs to the ischemic area and increasing host capillary density. These results provide for the first time direct evidence that OxLDL impaired the therapeutic potential of EPCs in ischemia-induced neovascularization through an inhibitory effect on the migration, adhesion, and incorporation of EPCs into vasculature and/or entrapment in the perivascular region in vivo. A therapeutic strategy based on SDF-1 administration ameliorated such defects and improved postischemic neovascularization.
The Journal of Lipid Research 04/2007; 48(3):518-27. · 5.56 Impact Factor
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ABSTRACT: To determine whether mobilized peripheral blood mononuclear cells (M-PBMNCs) obtained from patients with diabetes was impaired in therapeutic neovascularization in limb ischemia, and to explore the pathological mechanisms of the impairment.
Endothelial progenitor cells (EPC) were cultured in EGM-2MV, and then characterized by uptake of 1, 1-dioctadecyl-3, 3, 3, 3-tetramethylindocarbocyanine-labeled acetylated low density lipoprotein (Dil-AcLDL) and binding of ulex europaeus agglutinin (UEA). The number of EPC was compared between M-PBMNCs obtained from diabetic patients and those from normal subjects. M-PBMNCs obtained from diabetic patients, M-PBMNCs obtained from normal controls, or PBS were injected into the ischemic limbs of streptozotocin-induced diabetic nude mice. The limb blood perfusion was detected by laser Doppler blood perfusion imaging between these three groups in the following 1, 3, 7, 14, 21, and 28 days. Ambulatory score and ischemia damage were evaluated in the following 4 weeks. Capillary/fiber ratio was detected by CD31 or BS-1 lectin, and arteriole density was detected by alpha-smooth muscle actin (alpha-SMactin).
The number of EPC from diabetic patients were positively correlated with the blood perfusion (R = 0.486, P < 0.05) and capillary density (R = 0.491, P < 0.05), and the EPC number in diabetic patient were negatively correlation with their disease courses (R = - 0.587, P < 0.05). Transplantation of diabetic M-PBMNCs augmented the blood perfusion of ischemia hindlimbs, increased the capillary and arteriole densities, and promoted the collateral vessel formation. However, all the improvements were less significant in the diabetic patients than in the non-diabetic patients (P < 0.05).
Diabetes decreased the capability of M-PBMNCs to augment neovascularization in ischemia.
Zhongguo yi xue ke xue yuan xue bao. Acta Academiae Medicinae Sinicae 04/2007; 29(2):262-7.
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ABSTRACT: To explore the mechanism of the therapeutic efficiency of mobilized peripheral blood mononuclear cells (PBMNCs) with and without CD34+ cell depletion in ischemia nude mice.
After femoral ligation of mice, 1 x 10(6) PBMNCs, CD34+ cell depletion PBMNCs, or PBS were intramuscularly injected into the ischemic limb. Blood perfusion, ischemia damage, and capillary density of the limb were observed. VEGF expression in ischemic limbs was assayed by ELISA and immunohistochemistry.
PBMNCs transplant greatly improved the recovery of ischemic limbs. At day 28 after surgery, the blood perfusion rate of ischemic limbs recovered to (96.4 +/- 5.6)% from (20.3 +/- 4.2)% in PBMNCs transplanted group, compared with (71.3 +/- 4.4) % in PBS group (P <0.01). Depletion of CD34+ cells reduced the perfusion ratio to (83.8 +/- 5.2)% (P < 0.05). Capillary density in PBMNCs transplanted group was (521 +/- 47)/mm2, while in CD34+ cell-depleted group [ (396 +/- 21)/mm2] (P < 0.05). PBMNCs were found to incorporate into vascular network. VEGF was greatly up-regulated after transplantation of PBMNCs and was secreted in situ.
Transplantation of mobilized PBMNCs augments neovascularization in ischemic limb via supply of stem/progenitor cells and angiogenic factors. Depletion of CD34+ cells impaired therapeutic efficacy for limb ischemia.
Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 04/2007; 28(3):194-8.
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ABSTRACT: The limited ability of the heart to regenerate damaged tissue following a myocardial infarct results in progressive dysfunctions and consequently leads to heart failure. Cell therapy with stem cells for cardiac repair is emerging as an alternative strategy and demonstrates promising results. Recent advances suggest human umbilical cord may be a new source for stem cells. Human umbilical cords are easy to obtain and umbilical cord derived stem cells can be easily extracted and cryopreserved, allowing for individuals to store their own samples for possible future autologous use even if there were no immediate indication that stem cell therapy would be required. Therefore, we hypothesize that human umbilical cord derived stem cells may be the new cell source for the injured heart.
Medical Hypotheses 02/2007; 68(1):94-7. · 1.39 Impact Factor
