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The Effect of the Different Hemopoietic Microenvironment on the Differentiation of Hemopoietic Cells from Human Embryonic Stem Cells

Author: ZhaoHaiJun
Tutor: LuGuang
School: Central South University
Course: Stem Cell Engineering
Keywords: Embryonic stem cells Stromal cells Protein Hematopoietic
CLC: Q813
Type: Master's thesis
Year: 2007
Downloads: 81
Quote: 0
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Abstract


Objective To compare the yolk sac stromal cells from human fetal liver stromal cells and human fetal bone marrow stromal cells of human embryonic stem cells (human embryonic stem cells, hESC) difference to the efficiency of hematopoietic cell differentiation; proteomics screening to identify human fetal liver stromal cells and human fetal bone marrow stromal cells differentially expressed proteins, and to explore the possible mechanism of the stromal cells of human embryonic stem cells to hematopoietic cell differentiation efficiency difference. Materials and Methods induced using human embryonic stem cell lines as I built by the Department of the cell lines, and its the hematopoietic specific antigen (KDR, CD34, CD45) expression levels detected before induction. Ethics Committee, under the guidance of the Central South University, patients after obtaining informed consent, access to 4-6 weeks pregnant sac stromal cells isolated from the yolk sac; obtain embryonic bladders induction of labor in April to June, the separation of the fetal liver and bone marrow stromal cells. Before detection the three stromal cells of hematopoietic-specific antigen (KDR, CD34, CD45) expression levels. The first human embryonic stem cells spontaneously differentiate to form a 5-day EBs (embryonic bodies EBs), then the three different stromal cells as feeder cells, through the EB direct contact with five days of 10 days of culture, comparing different stromal cells of human embryonic stem cells to hematopoietic cell differentiation efficiency difference. Fetal liver stromal cells with 5 days of EB direct contact co-culture, observe the difference to the efficiency of hematopoietic cells of fetal liver stromal cells in direct contact with non-direct contact with five days of EB. The two-dimensional gel electrophoresis of human fetal liver stromal cells and human fetal bone marrow stromal cell protein profiles. Software analysis of differences in screening between proteins, part of the protein spots identified by mass spectrometry. Results 1. Three stromal cells were induced to use third-generation cell fusiform. Yolk sac stromal cells and bone marrow stromal cells do not express KDR, CD34, CD45; fetal liver stromal cells do not express the percentage of KDR, CD34 and CD45 expression were 0.30 ± 0.01%, 0.20 ± 0.01%. 2. Before the induction of hES not express KDR, CD34, CD45; expression of KDR spontaneous the 5d EB differentiation formed, CD34, CD45 percentage were 1.10 ± 0.01%, 0.30 ± 0.01%, 0.20 ± 0.02%. Different stromal cells induced 5 days EB to hematopoietic differentiation results are as follows: ① 5 days EB direct contact with the yolk sac stromal cells cultured for 10 days generated cells express KDR percentage of CD34, CD45, respectively, 1.80 ± 0.56%, 1.30 ± 0.14%, 1.05 ± 0.63 %; ② The 5 days of EB and fetal liver stromal cells in direct contact with cultured 10 days resulting cell expression for KDR, CD34, CD45 percentage was 34.0 ± 25.45%, 38.4 ± 24.8%, 72.6 ± 25.7%; ③ The 5 Tian EB and fetal liver stromal cell non-direct contact with cultured 10 days to generate the cells express KDR, CD34, CD45 percentage was 0.20 ± 0.10%, 0.37 ± 0.20%, 0.27 ± 0.14%; ④ 5 Tian EB with bone marrow stromal cells in direct contact with cultured 10 days generated cells express KDR, CD34, CD45 percentage was 2.50 ± 1.48%, 3.20 ± 0.56%, 1.65 ± 0.21%; ⑤ 5 days EB spontaneous differentiation 10 days generated cells express KDR, CD34, CD45 percentage was 0.30 ± 0.07%, 0.65 ± 0.07 %, 0.15 ± 0.07%. Efficiency significantly better than the other two stromal cells (P <0.05) compared with the spontaneous differentiation of 10 days, three types of stromal cells and five days EB direct contact, and are able to promote EB differentiation of hematopoietic cells and fetal liver stromal cells; fetal liver stromal cells in direct contact with the five days EB to culture significantly better than non-direct contact with the culture (P <0.05). 3. Using two-dimensional gel electrophoresis technique to establish the total protein map of human fetal liver stromal cells and human fetal bone marrow stromal cells. PDQUEST software analysis, screening in the stromal cells of human fetal liver expression of human fetal bone marrow stromal cells do not express the protein 30; 13 protein spots more than 10 times the amount of protein expression difference between the two stromal cells; 4 . Analysis of protein spots were identified by MALDI-TOF-TOF-MS 26, which protein is highly expressed in the stromal cells, including contraction and cell adhesion protein related protein and hematopoietic occurred signaling pathway in the fetal liver. Conclusions (1) and the yolk sac stromal cells and bone marrow stromal cells, stromal cells of the fetal liver is more conducive to human embryonic stem cells to hematopoietic cell differentiation; fetal liver stromal cells in direct contact with the five days EB culture is superior to non-direct contact with the culture. (2) analysis of identification discrepancy fetal liver stromal cells and human fetal bone marrow stromal cells between 26 protein spots. Which protein is highly expressed in the stromal cells, including cell contraction, adhesion ingredients hematopoietic occurred related signaling pathway protein in human fetal liver.

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