| Hematopoietic stem cells (HSCs) are the principal cell formation in humans and also it has been studied for longer times of all kinds of stem sells. The production of mature blood cells is a continual process that is the result of proliferation and differentiation of HSCs and committed progenitor cells, and differentiated cells. Hematopoietic cells have the potential for providing benefit in a variety of clinical settings. A number of malignancies can be successfully treated by high dose therapy /irradiation and haematopoietic progenitor cell transplantation (HPCT). However, thrombocytopenia and neutropenia always occur after the transplant and patients may require multiple platelet transfusions. It may be possible to reduce the period of thrombocytopenia by re-infusion of ex vivo expanded progenitors, derived from the progenitor cell graft. The limitation to many of these applications has been the total absolute number of defined target cells. Therefore many investigators have explored methods to culture hematopoietic cells in vitro to increase the numbers of these cells. Studies attempting to expand hematopoietic stem cells, progenitor cells, and mature cells in vitro have become possible over the past decade due to the availability of recombinant growth factors and cell selection technologes. Umblical cord blood has been demonstrated has more hematopoietic stem cells than peripherial blood and it has been used as an alternative source of bone marrow hematopoietic stem cells for transplantation in children. It is sufficient in our country. However, the insufficient number of cells in single umbilical cord blood limits its application for larger recipients and celltherapy. Thus, it is important to expand the committed progenitor cells for clinical use. To elucidate the capacity of different hematopoietic growth factors (HGFs) combinations for inducing HSC and HPC to proliferate and to differentiate different linages such as granulocytes, erythrocytes, megakaryocytes and dentritic cells in vitro, CD34+ cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS), and expanded with HGFs in a liquid culture system. Mononuclear cells isolated by routine method contain mature monocytes/macrophages, T/B lymphocytes and hematopoietic stem/progenitor cells. Eliminating a CD34 selection step not only avoids the cost and time associated with processing, but also the significant loss of up to 50% or more of CD34+ cells during the selection process. Potential benefits of selection, including removal of contaminating tumor cells or T-cells, may not be relevant in the CB setting. However, recent reports have suggested that mature and accessory cell populations present in a CB unit prior to selection may have negative effects on the ability of HSPC to proliferate ex vivo. Therefore, we established a new method for isolate the hematopoetic cells by using the character of the mature cells in the CB. First, we used the nylon fibre to delete the B-cells. Second the selected cells were cultured at plastic flasks at 37°C for 3 hours to eliminate the monocytes/macrophages. Cells were havested after each step for FACS analysis and culture assays. Results showed that cells selected by the method contain about 20% CD34+ cells and successfully expanded in liquid ulture system.We also have investigated the expansion of megakaryocyte progenitors from Lin-/CD126+ cells from CB. Lin-cells were purified by monoclonal antibody (include CD2,CD3,CD14,CD16,CD19,CD24,CD56,CD66b,CD41 and Glycophorin A) immunomagnetic selection and then selected by another immunomagnetic system using CD126 antibody. Lin-/CD126+ cells were cultured in serum free medium supplemented with TPO + SIL-6R + IL-6 + SCF for 14 days. After this time, proliferation of the cultured cells was assessed by flow cytometry using lineage specific markers (CD41 and CD61). It was possible to significantly expand the number of CD41+/CD61+ cells derived from CB (20.66 folds at 7days and 39.55 folds at 14days; n=6). To evaluate methods for inducing hematopoietic stem/progenitor cells to differentiate to dendritic cells (DCs), granulocytes and erthrocytes, different combination of HGFs, including SCF, IL-3, IL-4, GM-CSF, TNF-α, IL-6, EPO and FL were studied. Also, mixed CB plasma was used to replace the animal serum. FL+GM-CSF+TNF-α+IL-4+SCF combination was used for dentritic cells differentiation. Cells harvested after 14 days were detected for morphological and phenotypic analysis by microscope, and FACS respectively. After 14 days of liquid culture, the induced cells have the typic morphology and high expression of CD1a, HLA-DR and CD80. (SCF+IL-3+IL-6+G-CSF) and (SCF+IL-3+IL-6+EPO) HGFs combinations were used to differentiate granulocytes and erthrocytes, respectively. The expended cell number and the ratio of the alive cells, the differentiation efficiency were detected during culture by using cell number counting, typan blue staining and flow cytometry analysis. The combination of HGFs increased the cell number by about 1006.4±103.2 folds in granulocytes lineage and about 1028.6±228.4 folds in erthrocytes lineage. FACS analysis showed that the about 80% cells expressing the myeloid marker CD11b and about 60% cells expressing the erthrocytic markerGlycophorin A. The growth peak is at 14 days of culture in both lineages and decreased at about 33 days of culture. Safety evaluations were detected during culture by using chromosome analysis, apoptosis related molecular analysis and nude mice injection analysis, respectively. No abnormity was found in the chromosome analysis of expanded cells and no tumor was found in the nude mice after 35 days of expanded cells injection. The Fas receptor and the expanded cells have the ability of licking up germ. Our results indicated that freshly isolated CD34+ cells did not express Fas Ag and CD34+ cells cultured with hematopoietic growth factors gradually became positive for Fas Ag. Caspase-3 protein expressed at a very low level in freshly isolated CD34+ cells and it was upregulated when the cells were expanded in suspension culture with growth factors for 3 days. Telomerase analysis also indicated that the cells expanded in vitro did not have the character of eternality. We concluded that using different combinations of HGFs and liquid culture system can induce hematopoietic stem/progenitor cells derived from CB into different lineages of blood cells. The cells number was significantly expanded and did not have the character of eternality. It may be possible to use ex vivo expanded progenitors and blood cells for clinical trial.
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