The Amplification Effect Of Hematopoietic Stem/Progenitor Cells Co-cultured With Mesenchymal Stem Cells And Endothelial Progenitor Cells In Vitro

hematopoietic stem/progenitor cells co-cultured with mesenchymal stem cells and withendothelial progenitor cells in vitro.Methods: Isolate MSCs from normal newborn umbilical cord, and identify the cellsfrom immune phenotype CD14, CD29, CD31, CD34, CD44, CD45, CD73, HLA-ABC andthe potential of differentiating to fat cells and osteoblasts. Identify EPCs from immunephenotype CD31, CD34, CD133, CD146. MSCs and EPCs were treated with Mitomycin Cand prepared to cytotrophoblast respectively. The experiment has three groups in total:(1)control(non-cytotrophoblast);(2)MSC group(MSCs as the cytotrophoblast);(3)EPCgroup(EPCs as the cytotrophoblast).Isolate mononuclear cells(MNCs) from normal newborn umbilical cord blood, andanalyze the proportion of HSCs/HPCs by flow cytometry. Then place the same number ofMNCs on the MSCs and EPCs cytotrophoblast respectively, or only on the culture medium,in order to observe the changes of MNC number on the4~th and the7~th day in three groups.The same number of MNCs which are before amplification, on the4~th day and the7~th day ofco-culturing are seeded in methylcellulose medium. After7to10days, we observe thecolony volume formed by HSCs/HPCs, and compare the number of colony. Analyze theexpression of CD34on HSCs/HPCs on the4~th and the7~th day, and detect the proliferation ofMNCs in each group on the first, third,5~th,7~th day by flow cytometry. Collect the culturedsupernatant of MSCs and EPCs after24hours of being treated by Mitomycin C, then detectand compare the levels of IL-3, IL-6, SCF, TPO, Flt3L and VEGF by ELISA assay.Results:1. On the4~th and the7~th day of co-culturing, the results from microscopy and trypanblue staining counting show that the expansion folders of MNCs co-cultured with MSCs orEPCs are more than MNCs only in culture medium obviously, especially MNCs on EPCs.On the4~th day of co-culturing, the number of MNCs in MSC group is1.71-fold than control（**，p<0.01）,and that in EPC group is about2.93-fold than control（*，p<0.05）. On the7~th day of co-culturing, the number of MNCs in MSC group is1.38-fold than control（*， p<0.05）, and that in EPC group is about2.10-fold than contro（l**，p<0.01）. Moreover, thenumber of MNCs in EPC group is approximately1.53-fold than that in MSC group（**，p<0.01）.2. After7days’ co-culturing, the expression of CD34on HSCs/HPCs is detected byflow cytometry, and the result shows that the proportion of CD34+CD45-HSCs/HPCs inthree groups all declines, especially in EPC group. On the contrary, the declining extent ofMSC group is the least obvious. CD34+CD45-HSCs/HPCs account for2.20%in MNCsbefore amplification. After7days, CD34+CD45-HSCs/HPCs proportion changes to1.40%incontrol. And that in MSC group declines to1.85%, likewise, the proportion in EPC group is0.41%.3. The results of colony forming experiment show that the total number and the volumeof colony in MSC group are more than other two groups.7to10days after seedingHSCs/HPCs which have been co-cultured for4days in methylcellulose medium, the totalnumber of colony in MSC group is2.47-folds than that in EPC group（**，p<0.01）. Andsimilarly,7to10days after seeding HSCs/HPCs which have been co-cultured for7days inmethylcellulose medium, the same item in MSC group has already reached3.45-folds thanthat in EPC group（**，p<0.01）.However, there are no differences between control and EPCgroup significantly.4. The proliferation of MNCs is analyzed by flow cytometry, and the result shows thatthe CFSE fluorescence intensity of MSC group and EPC group decreased more obviouslycompared with control on the first, third,5~th,7~th day, especially the EPC group.5. Levels of six cytokines in cultured supernatant are detected by ELISA assay. Theresults show that the differences between levels of IL-3and Flt3L in three groups are notstatistical; however, the level of IL-6in MSC group is significantly higher than control（p<0.01） and that in EPC group（p<0.01）; the level of SCF in EPC group is higher thancontrol and that in MSC group（p<0.01）; the level of TPO in EPC group is higher thancontrol（p<0.01）; and the level of VEGF in MSC group and EPC group are both higher thancontrol（p<0.01）.Conclusions: Compared with EPCs, MSCs can be a more suitable nourishing layer forHSC/HPCs in vitro. MSCs can maintain the expression of CD34, inhibit differentiation andretain the hematopoietic reconstitution and homing ability of HSCs/HPCs. The effect may be related with IL-6. But EPCs can accelerate the differentiation to mature blood cells ofHSCs/HPCs effectively, and this may have some specific relationship with TPO.