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ABSTRACT Background: Hematopoietic stem/progenitor cell transplantation is the main treatment option for hematological malignancies and disorders. One strategy to solve the problem of low stem cell doses used in transplantation is pre-transplant expansion. We hypothesized that using fibronectin-coated microfluidic channels would expand HSPCs and keep self-renewal potential in a three-dimensional environment, compared to the conventional method. We also compared stem cell homing factors expression in microfluidic to conventional cultures. Materials and methods: A microfluidic device was created and characterized by scanning electron microscopy. The CD133+ cells were collected from cord blood and purified. They were subsequently cultured in 24-well plates and microfluidic bioreactor systems using the StemSpan serum-free medium. Eventually, we analyzed cell surface expression levels of the CXCR4 molecule and CXCR4 mRNA expression in CD133+ cells cultured in different systems. Results: The expansion results showed significant improvement in CD133+ cell expansion in the microfluidic system than the conventional method. The median expression of the CXCR4 in the expanded cell was lower in the conventional system than in the microfluidic system. The CXCR4 gene expression up-regulated in the microfluidic system. Conclusion: Utilizing microfluidic systems to expand desired cells effectively is the next step in cell culture. Comparative gene expression profiling provides a glimpse of the effects of culture microenvironments on the genetic program of HSCs grown in different systems.
Subject(s)
Fibronectins , Hematologic Diseases , Neoplastic Stem Cells , Hematopoietic Stem Cells , Hematologic Neoplasms , Bioreactors , Receptors, CXCR4 , Fetal BloodABSTRACT
Objective:To observe the effect of intra-bone marrow injection (IBMI) of fetal liver mononuclear cell suspension on reconstructing the hematopoietic system in mice. Methods : The fetal liver mononuclear cells from the mice with gestational age of 13.5 days were obtained by Ficoll separation technique, and then the hematopoietic stem cells (HSCs) were obtained by magnetic bead sorting. The fetal liver mononuclear cells were injected into the tibias of 137Cs irradiated mice by IBMI. The recipient mice were observed for reconstitution of the hematopoietic system by routine blood examination, blood smear and bone marrow smear. The donor HSCs in the bone marrow of the recipient mice was subjected to flow cytometry 1 year after transplantation. Results: HSCs accounted for 0.171% in the mouse fetal liver mononuclear cells. One week after transplantation of mouse fetal liver mononuclear cells, blood cells derived from donor cells were detected from peripheral blood; in the third week after transplantation, peripheral blood leukocytes, red blood cells and hemoglobin of recipient mice were restored to the pre-irradiation levels; the percentage of donor-derived blood cells in peripheral blood in the fifth week was stable; the HSCs in the bone marrow of recipient mice were derived from donor cells 1 year after transplantation. Conclusion: The murine fetal liver mononuclear cells can efficiently reconstruct the hematopoietic system of mice by IBMI.
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Objective • To explore the differences in morphology, molecular characteristics and biological functions of different types of mouse fetal liver stromal cells. Methods • E13.5 mouse fetal liver stromal cells were obtained by adherent culture, and different cell types were distinguished by morphology and expression of surface markers, such as CD44, CD29, CD106, CD45 and Sca-1. Then microarray assay was conducted by Mouse Genome 430 2.0 Array and analyzed at P3 or <-2 to identify differential expression genes. Canonical pathway and biological function analyses were performed by using ingenuity pathway analysis (IPA software). Results • Spindle-like (CD45+CD106-CD29+CD44+Sca-1-) and fibroblast-like (CD45-CD106+CD29+CD44+Sca-1-) fetal liver stromal cells were isolated in this study according to the cell morphology. The 1 485 highly-expressed genes in spindle-like cells mainly involved in immune and inflammation-related signaling pathways; while the 3 374 highly-expressed genes in fibroblast-like cells mainly involved in extracellular matrix formation and cellular adhesion. Conclusion • Mouse fetal liver stromal cells have strong heterogeneity in biological characteristics and functions, especially in hematopoietic promoting capability.
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A double-copy Moloney leukemia virus-based retroviral vector containing both the Neo~(R) gene and a mutant human dihydrofolate re-ductase(S31 mutation) cDNA was packaged into the Amphotropic packaging cell hne GP-EAM12( AM12), and a Amphotropic producer cell hne (named AM12-S31)was obtained. In this study, we investigated its drug resistant characteristics, viral titer and for murine hematopoietic progenitor cells transduction as well. MTT assay verified that the AM12-S31 cells were resistant to G418 and methotrexate(MTX), the IC50 were more than 800 ?g/ml and 100 ?M respectively while the control cell line AM12 was sensitive to both drugs, the IC50 were 180 ?g/ml and 10 ?M, respectively. The viral titer for this cell line was approximately 7.8? 104~4.2? 105 G418-resistant colony forming units/ml. The replication-competent virus can not be detected in this producer cell line. We also use the AM12-S31 cells to transfect murine hematopoietic cells (By coculture) . The positive colonies were found in all the G418 concentrations using CFU-GM assay. No G418-resistant colony was found using AM12 transfection. The infected murine marrow cells were returned to lethally irradiated(900rad)recipients. The murine transplanted with AM12-S31 infected marrow cells showed protection from lethal MTX toxicity as compared with AM12 infected animals. Evidence for integration and the proviral DNA was obtained by PCR amplification of proviral DNA. These results indicated this producer cell hne could produce high titer, high-efficiency and non-replcational competent virus. The murine marrow cells could be transfected successfully using this system, and express the foreign gene. The lethal irradiated murine marrow function could be reinstitution by infusing the hematopoietic progenitor cells tranducted with human mutant dihydrofolate reductase. In my opinion, this system would play an important role in research the long-term protection of murine marrow hematopoietic function and drug resistant gene therapy.