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ABSTRACT: In order to investigate the effect of mesenchymal stem/progenitor cells on proliferation and differentiation towards megakaryocytes of CD34+ cells from human umbilical cord blood in vitro.
After mesenchymal stem/progenitor cells were advancely planted in DMEM medium and grown up to 80%, then the CD34+ cells were added to culture with mesenchymal stem/ progenitor cells or without mesenchymal stem/progenitor cells in DMEM for 14 days with TPO + IL-3 + SCF, TPO + IL-3 + SCF + IL-11 respectively. After cultured for 14 days, mononuclear cells (MNCs) were counted by automatic cell analyzer. The number of CD41+ cells and platelets were detected by flow cytometry. Platelets function were assessed through platelet aggregation test which was induced by thrombin.
As compared with the control group, the number of MNCs of co-culture system was not increased significantly (P > 0.05), but the number of CD4+ cells and platelets were increased significantly (P < 0.05). The platelets were aggregated by thrombin induced which could be seen in microscope or flow cytometry.
It is concluded that mesenchymal stem/progenitor cells may be promoted to induce the cord blood CD34+ cells to differentiate towards megakaryocyte in the culture medium.
Zhongguo ying yong sheng li xue za zhi = Zhongguo yingyong shenglixue zazhi = Chinese journal of applied physiology 02/2008; 24(1):77-80.
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ABSTRACT: The aim of this study was to investigate the feasibility of the human cord blood adult stem cells (ASCs) to differentiate into hepatocytes in vitro induced by combined stimulation with hepatocyte growth factor (HGF), stem cell factor (SCF) and leukemia inhibitory factor (LIF). The adult stem cells were obtained through density gradient centrifugation and magnetic activated cell sorting (MACS). The adult stem cells were cultured in DMEM with HGF (10 ng/ml)+SCF (10 ng/ml)+LIF (10 ng/ml) in induced group I. In induced group II the enriched cells were cultured in DMEM with SCF (10 ng/ml)+LIF (10 ng/ml) and the undifferentiated cells acted as the control group without the factors. The morphology of cells was observed by the inverted phase contrast microscopy; the expression of albumin (Alb), human hepatocyte cytokeratin (CK18) and alpha-fetoprotein (AFP) were detected by immunofluorescence, immunohistochemistry and RT-PCR assay in the 21-day culture. Alb secreted by hepatocytes in the medium was determined by radioimmunoassay (RIA) at day 7, 14, 21, 23 and 25. The results showed that the shapes of ASCs changed and their sizes and number increased in the course of culture in group I. After being induced for three weeks, the cells turned round and resembled hepatocyte-like cells. The mRNA for Alb could be detected by RT-PCR in the differentiated adult stem cells in group I, and the mRNA for AFP was poorly detected by RT-PCR at day 21. Alb and CK18 were positive through immunofluorescence and immunohistochemistry at day 21, compared with group II and the control group. In group I, Alb in the medium significantly increased, compared with control group, and reached the highest level at day 21, then decreased at day 23. It is concluded that under some definite inducing conditions, human cord blood adult stem cells can differentiate into hepatocyte-like cells and HGF plays a critical role during the course.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 01/2008; 15(6):1220-5.
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ABSTRACT: This study aimed to investigate the potentials of ex vivo expansion and pluridifferentiation of cryopreserved adult human bone marrow mesenchymal stem cells (hMSCs) into adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced to adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and expression of triglyceride or neuron-specific enolase and nestin was detected. The result showed that the resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and insulin-like growth factor I (IGF-I) showed adipogenesis, and lipid vacuole accumulation was detectable after 21days. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron-specific enolase (NSE), which are special surface markers associated with neural cells at different stages. This study suggested that resuscitated hMSCs should still be a population of pluripotential cells and should be accessible for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.
Cell Biology International 06/2007; 31(5):444-50. · 1.48 Impact Factor
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ABSTRACT: This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells (hMSCs) into chondrocytes, adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced into chondrocytes, adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and detected for expressions of type II collagen, triglyceride or neuron-specific enolase and nestin. The result showed that the resuscitated cells could differentiate into chondrocytes after exposure to transforming growth factor beta(1) (TGF-beta(1)), insulin-like growth factor I (IGF-I) and vitamin C (V(C)), and uniformly changed morphologically from a spindle-like fibroblastic appearance to a polygonal shape in three weeks. The induced cells were heterochromatic to safranin O and expressed cartilage matrix-procollagenal (II) mRNA. The resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin and IGF-I showed adipogenesis, and lipid vacuoles accumulation was detectable after 21 d. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron specific endolase (NSE) that were special surface markers associated with neural cells at different stage. This study suggested that the resuscitated hMSCs should be still a population of pluripotential cells and that it could be used for establishing an abundant hMSC reservoir for further experiment and treatment of various clinical diseases.
Journal of Zhejiang University SCIENCE B 03/2007; 8(2):136-46. · 1.10 Impact Factor
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ABSTRACT: A novel indirect co-culture system was established to support ex vivo expansion of hematopoietic progenitors in umbilical cord blood (UCB) by using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human-marrow-derived mesenchymal stem cells (tfhMSCs) as a feeder. UCB CD34+ cells were isolated and cultured by using five culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were taken to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient mouse (SCID) repopulating cell (SRC) assay was performed to confirm the ability of the indirect co-cultured cells from the tfhMSCs system to reconstitute long-term hematopoiesis. Results showed significant differences in the number of total nucleated cells (TNCs) among the culture systems with respect to serum-containing medium or serum-free medium during 14-day culture. In addition, on day 14, the outputs of CD34+ cells, the colony-forming units (CFUs) in culture, and the CFUs in mixed colonies containing erythroid and myeloid cells and megakaryocytes in the tfhMSC indirect co-culture system were significantly enhanced. The LTC-IC assay demonstrated that the tfhMSCs indirect co-culture system had the strongest activity. The SCID-SRC assay confirmed the extensive ability of the expanded cells from the tfhMSCs indirect co-culture systems to reconstitute long-term hematopoiesis. Furthermore, polymerase chain reaction analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of non-obese diabetic/SCID mice. Thus, hMSCs transduced with TPO/FL, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro. The tfhMSC indirect co-culture system may therefore be a suitable system for ex vivo manipulation of primitive progenitor cells under non-contact culture conditions.
Cell and Tissue Research 11/2006; 326(1):101-10. · 3.11 Impact Factor
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ABSTRACT: To establish a novel coculture system for ex vivo expansion of umbilical cord blood(UCB) hematopoietic progenitors using thrombopoietin (TPO)/Flt-3 ligand (FL)-transduced human marrow-derived mesenchymal stem cells (tfhMSCs) as feeder.
UCB CD34+ cells were isolated and cultured using four culture systems in serum-containing or serum-free medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture (LTC-IC) output. Finally, the severe-combined immunodeficient (SCID) mouse-repopulating cell (SRC) assay was performed to confirm ability of the cultured cells to reconstitute long-term hematopoiesis.
There were no significant differences in the number of total nucleated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34+ cells, CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient (SCID) mouse repopulating cell (SRC) assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice.
The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.
World Journal of Gastroenterology 02/2006; 12(3):393-402. · 2.47 Impact Factor
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ABSTRACT: To explore the biological characteristics of mesenchymal stem cells (MSC) derived from umbilical cord blood (UCB) and their supporting capacities in ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs).
Low-density mononuclear cells (MNCs) from UCB were cultured in IMDM containing 20% FBS to form confluent adherent cells through 15 passages. Some cytokines in the conditioned medium were determined with ELISA. UCB-derived adherent cells were displayed with antibodies and analyzed with flow cytometry. The supporting capacity of UCB-derived adherent cells for ex vivo expansion of CD34(+) cells was assayed by co-culture in a two step culture. UCB-derived adherent cells were induced for chondrogenic differentiation with chondrogenic medium, and the induced cells were analyzed for the type II pro-collagen gene expression with RT-PCR.
The mean number of adherent fibroblast like colonies derived from UCB was (3.5 +/- 0.7)/10(6) MNCs. UCB-derived MSCs could survive for at least 15 passages of expansion. In their undifferentiated status, UCB-derived MSCs were CD13(+), CD29(+), CD90(+), CD105(+), CD166(+), SH2(+), SH3(+), SH4(+), CD45(-), CD34(-), and CD14(-). Stem cell factor (SCF), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) could be detected in the supernatant of the cultures. The MSCs cultured in chondrogenic media could differentiate into chondrogenic cells and express type II pro-collagen mRNA. UCB-derived MSCs could support the proliferation and differentiation of UCB CD34(+) cells in vitro.
UCB-derived MSCs are similar to those derived from adult bone marrow and can support the proliferation of hematopoietic stem/progenitor cells.
Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi 03/2005; 26(2):65-8.
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Zhongguo Zhong xi yi jie he za zhi Zhongguo Zhongxiyi jiehe zazhi = Chinese journal of integrated traditional and Western medicine / Zhongguo Zhong xi yi jie he xue hui, Zhongguo Zhong yi yan jiu yuan zhu ban 08/2004; 24(7):666-9.
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ABSTRACT: Human mesenchymal stem/progenitor cells (MSPC) ar pluripotent, being the precursors for marrow stroma, bone, cartilage, muscle and connective tissues. Although the presence of hematopoietic stem/progenitor cells (HSPC) in umbilical cord blood (UCB) is well known, that of MSPC has been not fully evaluated.
In this study, we examined the immunophenotype, the supporting function in relation to ex vivo expansion of hematopoietic stem progenitor cells and the chondrogenic differentiation of cultured cells with characteristics of MSPC from UCB. When UCB nucleated cells were isolated and 107 cells cultured in IMDM with 20% fetal bovine serum, the mean number of adherent fibroblastlike colonies was 3.5+/-0.7/10(6) monuclear cells.
UCB-derived MSPC could be expanded for at least 15 passages. In their undifferentiated state, UCB-derived MSPC were CD13(+), CD29(+), CD90(+), CD105(+), CD166(+), SH2(+), SH3(+), SH4(+), CD45(-), CD34(-), and CD14(-); they produced stem cell factor, interleukin 6 and tumor necrosis factor alpha. UCB-derived MSPC cultured in chondrogenic media differentiated into chondrogenic cells. UCB-derived MSPC supported the proliferation and differentiation of CD34(+) cells from UCB in vitro.
UCB-derived MSPC have the potential to support ex vivo expansion of HSPC and chondrogenic differentiation. UCB should not be regarded as medical waste. It can serve as an alternative source of mesenchymal stem cells and may provide a unique source of fetal cells for cellular and gene therapy.
Haematologica 08/2004; 89(7):837-44. · 6.42 Impact Factor
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ABSTRACT: Vaccinia virus genes A18 and G2 affect the elongation and termination of postreplicative viral gene transcription in opposite ways. Viruses with mutations in gene A18 produce abnormally long transcripts, indicating that A18 is a negative transcription elongation factor. Viruses containing mutations in gene G2 produce transcripts that are abnormally short, truncated specifically from their 3′ ends, indicating that G2 is a positive transcription elongation factor. Despite the fact that both A18 and G2 are essential genes, A18-G2 double-mutant viruses are viable, presumably because the effects of the mutations are mutually compensatory. In addition, the anti-poxviral drug isatin-β-thiosemicarbazone (IBT) seems to enhance elongation during a vaccinia infection: IBT treatment of a wildtype vaccinia infection induces a phenotype identical to an A18 mutant infection, and G2 mutant viruses are dependent on IBT for growth, presumably because IBT restores the G2 mutant truncated transcripts to a normal length. These observations inspire two independent genetic selections that have now been used to identify an additional vaccinia gene, J3, that regulates postreplicative transcription elongation. In the first selection, a single virus that contains an extragenic suppressor of the A18 temperature-sensitive mutant, Cts23, was isolated. In the second selection, several spontaneous IBT-dependent (IBTd) mutant viruses were isolated and characterized genetically. Marker rescue mapping and DNA sequence analysis show that the extragenic suppressor of Cts23 contains a point mutation in the J3 gene, while each of seven new IBTd mutants contains null mutations in the J3 gene. The J3 protein has previously been identified as a (nucleoside-2′-O-)-methyltransferase and as a processivity subunit for the heterodimeric viral poly(A) polymerase. The nature of the two independent selections used to isolate the J3 mutants strongly suggests that the J3 protein serves as a positive postreplicative transcription elongation factor during a normal virus infection.
Virology.
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ABSTRACT: Ghrelin is a 28-amino-acid peptide secreted predominantly by X/A-like cells of the gastric fundus. Ghrelin increases pancreatic β-cell proliferation and survival via sequential activation of phosphatidylinositol-3 kinase (PI3K) and Akt. The transcription regulator Foxo1 is a prominent effector of PI3K/Akt; when it is inhibited, pancreatic β-cells are protected against fatty-acid-induced apoptosis. We investigated the role of Foxo1 in the protective effect of ghrelin under lipotoxic conditions in the MIN6 pancreatic β-cell line. Results showed that ghrelin promoted cell proliferation and attenuated palmitate-induced apoptosis in cultured MIN6 cells. Nuclear exclusion of Foxo1 was necessary for the function of ghrelin. Treatment of MIN6 cells with palmitate and ghrelin-induced rapid nuclear exclusion and phosphorylation of Foxo1. Unlike the JNK inhibitor SP600125, Akt inhibitor IV blocked the anti-lipotoxic effect of ghrelin and stimulated Foxo1 nuclear translocation. In addition, treatment with ghrelin combined with SP600125 showed a synergistic antiapoptotic effect in palmitate-treated MIN6 cells. Ghrelin also inhibited the endoplasmic reticulum stress pathway of apoptosis in MIN6 cells, decreased expression of cytoplasmic triglyceride, and downregulated gene expression of Bcl-2-associated X (BAX), sterol-response element-binding protein 1c (SREBP1c), and C/EBP homologous protein (CHOP-10). These findings suggest that ghrelin protects pancreatic β-cells from lipotoxicity by inhibiting the nuclear translocation of Foxo1.
Peptides.
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ABSTRACT: Prior genetic analysis suggests that the vaccinia virus J3 gene product, previously characterized as a bifunctional (nucleoside-2′-O-)-methyltransferase and poly(A) polymerase stimulatory factor, is a postreplicative positive transcription elongation factor. To test this hypothesis, viruses bearing mutations in the J3 gene were characterized with respect to viral protein and RNA synthesis in infected cells. The analysis reveals that compared to wt virus infections, J3 mutants synthesize reduced amounts of large late viral proteins and shorter-than-normal intermediate and late mRNAs. Structural analysis of one late mRNA shows that it is specifically truncated from the 3′ end, thus accounting for its shorter than normal chain length. Thus J3 mutant viruses are defective in elongation of transcription of postreplicative viral genes, strongly suggesting that the J3 gene product normally acts as a positive transcription elongation factor. Biochemical analysis of one J3 missense mutant demonstrates that it retains poly(A) stimulatory activity but is defective in (nucleoside-2′-O-)-methyltransferase activity. Thus the elongation factor activity of the J3 gene product is independent of the poly(A) stimulatory activity. It remains to be determined whether the (nucleoside-2′-O-)-methyltransferase and elongation factor activities of the J3 protein are linked or can be uncoupled by mutation.
Virology.
