| Object Allogeneic hematopoietic stem cell transplantation is one of the best methods to treat hematological malignancies and kinds of immune disorders. With the development of cell therapy and HSCT, BMHSC is gradually replaced by GPBSC because of its more convenient collection and less invasion, which is now the main cell source of HSCT and cell therapy. The deficiency of cell number is still the most stubborn obstruction in allo-HSCT. This research aims at seeking a safely effective way to expand hematopoietic stem cells ex vivo in a shorter time, so that we can obtain a fine cell product for potential clinical use.Contents 1. Extract microvesicles from bone marrow mesenchymal stem cells culture supernatant. Identify MVs from three aspects, namely morphological character, protein content and surface markers.2. Culture GPBMC with MSC-MVs to see the influence on hematopoietic proliferation.3. Based on the previous experiments, we added SR-1, an aryl hydrocarbon receptor, into the culture system to see the influence on hematopoietic proliferation.4. Preliminary studied the security and effectiveness in mice with the products of cultured cells.Methods 1. Extract MVs from cultured MSCs supernatant by multi-step differential velocity centrifugation. Identify MVs by electron microscope analysis, Micro-BCA protein contents measurement and FCS surface antigen detection.2.The experiment was divided into two groups. MV groups(MV) with 10μg/mL MVs and control group(CC) with same volume of PBS. The proliferation of GPBMC was evacuated by cell counting, flow cytometry, colony culture and karyotype analysis technique.3. Based on the previous study, we added SR-1 into the culture system. To culture GPBMC as follows:CC group and MV groups as described before, SR-1 group(SR-1)with 0.75μM SR-1 and mixed group(M+S) with lOμg/mL MVs and 0.75μM SR-1.The proliferation of GPBMC was evacuated by cell counting, flow cytometry, colony culture and karyotype analysis technique.4. Flow sorting CD34+ cells from chosen groups and fresh samples. Infuse 1×105 CD34+cells into NPG mice with 2Gy TBI. Observe the chimeric rate by flow cytometry.Results 1.MSC-MVs are 20nm-100nm circular or cup-shaped vesicles under electron microscope observation. Every 60mL supernatant could extract about 60μg protein. The flow cytometry shows that CD63 and CD44 are positive with a rate of 96.0% and 50.2%, respectively, while HLA-DR,CD34 and CD29 etc. are negative.2. MSC-MVs has an influence on proliferation of GPBMC. The number increases as culture time rises. When cultured 2 days, the cell number of MV groups is 1.49±0.15 fold of control group(P>0.05).When cultured 4 days, the cell number of MV groups is 2.20±0.24 fold of control group (P<0.05).When cultured 6 days, the cell number of MV groups is 2.37±0.21 fold of control group(P>0.05).Combined the cell amount and the proportion of CD34+, we figured that the CD34+cell number of MV groups is 1.76±0.30 fold of control group after 2 days culture and 1.95±0.20 fold after 4 days culture. Besides, cells from MV groups are capable of colony formation and do not show any chromosome abnormality.3.There is no significant difference between the cell number of SR-1 group and control group. The cell number of mixed group is 1.42±0.22 fold of control group after 4 days culture. The number of CD34+cell of control group and SR-1 groups are of no difference. The cell number of M+S group is lower than MV group while the CD34+cell number is higher in both 2d and 4d.4. The results show that there is no significant difference between two groups and the chimeric rate are above 8% after 1 month.Conclusions We could successfully extract MVs from MSC culture supernatant by multi-step differential velocity centrifugation. MSC-MVs can promote HSC expansion. The combination of SR-1 and MSC-MVs is better for keeping HSC sternness. |
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