| Object To observe the inductive efficiency of deriving hematopoietic cells from human embryonic stem (hES) cells co-cultured with human yolk sac stromal cells,fetal liver stromal cells or fetal bone marrow stromal cells. In order to investigate the mechanism of the different inductive efficiency of the two stromal cells, 2-DE maps of fetal liver stromal cells and fetal bone marrow stromal cells were established and partially differentially expressed proteins were identified..Material & Methods The hES cell line was established in our laboratory. Human yolk sac stromal cells were isolated from human yolk sac of 4-6 weeks and fetal liver stromal cells or fetal bone marrow stromal cells were isolated from abort fetus of 4-6 months. All samples were obtained under informed consent using forms approved by Central South University Ethic Investigation Committee. Exclusive surface antigens of hematopoiesis were detected on three stromal cells and hES before induction.Firstly the day-5 embryonic bodies (5dEBs) were formed by the hES cells spontaneous differentiation. Then the 5dEBs cells were induced into hematopoietic stem cells by co-culture with human yolk sac stromal cells,fetal liver stromal cells or fetal bone marrow stromal cells for 10 days respectively . The effect of directly contact between fetal liver srromal cells and 5d EBs on the inductive efficiency was discussed.Using 2-DE technology, the total proteins of fetal liver stromal cells and fetal bone marrow stromal cells were separated and 2-DE maps were established. MALDI-TOF-TOF-MS was used to analyze partial differentially expressed protein spots and get peptide mass fingerprinting and MS/MS .Finally ExPASY database were used for protein identification.Results1) Exclusive surface antigens of hematopoiesis KDR,CD34,CD45 were not detected on Human yolk sac stromal cells and fetal bone marrow stromal cells. Percentage of CD34,CD45 positive cells among fetal liver stromal cells was 0.30±0.02%,0.20±0.01% respectively.2) Human embryonic stem (hES) cells didn't express surface antigen KDR,CD34,CD45; the day-5 embryonic bodies (5dEBs) formed by the hES cells spontaneous differentiation expressed surface antigen KDR 1.10+0.01%> CD34 0.30±0.01%,CD45 0.02±0.02%,; The results of flow cytometry analysis demonstrated that the population of the cells co-cultured with human yolk sac stromal cells contained KDR 1.80±0.56%,CD34 1.30±0.14% or CD45 1.05±0.63% positive cells; the population of the cells co-cultured with human fetal liver stromal cells contained KDR 34.00±25.45%,CD34 38.40±24.80% and CD45 72.60±25.70% positive cells; the population of the cells co-cultured without contact with human fetal liver stromal cells contained KDR 0.20±0.10%, CD34 0.37±0.20% and CD45 0.27 + 0.14% positive cells; the population of the cells co-cultured with human fetal bone marrow stromal cells contained KDR 2.50±1.48%, CD34 3.20±0.56% and CD45 1.65±0.21% positive cells. Compared with spontaneous differentiation of EBs, all of three stromal cells could induce EBs into hematopoietic cells (p < 0.05) . The inductive efficiency of deriving hematopoietic cells from EBs co-cultured in contact with human fetal liver stromal cell was higher than EBs co-cultured without contact with human fetal liver stromal cell (p < 0.05).3) 2-DE patterns of fetal bone marrow stromal cells and fetal liver stromal cells were obtained. Analyzed with PDQUEST software, 30 protein spots were detected in Gel image of fetal liver stromal cells, but not detected in Gel image of fetal bone marrow stromal cells; the number of differentially expressed protein spots above 10-fold between them is 12.4) Using MALDI-TOF-TOF-MS,26 protein spots were identified, highly expressed proteins in fetal liver stromal cells including proteins related to cell movement and adhesion and Serine-threonine kinase receptor-associated protein, which inhibited TGF-βsignal transduction..Conclusions (1) Compared to human yolk sac stromal cells and fetal bone marrow stromal cells, fetal liver stromal cells are more efficient to induce human embryonic stem (hES) cells into hematopoietic cells; Compared to co-culture without contact, co-culture in contact with fetal liver stromal cell was superior to induce ESC into hematpoietic cell.(2) In this study, MALDI-TOF-TOF-MS was used to identify partial differentially expressed protein spots between fetal liver stromal cells and fetal bone marrow stromal cells. Some of these highly expressed proteins in fetal liver stromal cells were related to cell movement and adhesion and Serine-Threonine Kinase Receptor-associated Protein (STRAP).
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