Abstract:

BACKGROUND: Further studies are needed to understand the cytobiological character, functional regulation, gene changes and expression difference of CD34⁺ and CD133⁺ stem cells induced by basic fibroblast growth factor (bFGF) using gene chip.

OBJECTIVE: To compare the differences of gene expression and the response to bFGF of human umbilical cord CD34⁺ and CD133⁺ cells, and to explore gene expression changes of bFGF-induced umbilical cord CD34⁺ and CD133⁺ hematopotic stem cells/hemapoietic progenitor cells in vitro.

METHODS: Human umbilical cord blood CD34⁺ and CD133⁺ cells were isolated and purified by MiniMACS immunomagnetic beads selection. The CD34⁺ and CD133⁺cells were cultured for 10 to 15 days in DMEM/F12 medium, supplemented with bFGF and B27. Total RNA from these cells was extracted and the genetic level of these cells was performed using Oligo GEArray® chip and GEArray software. Selected rate of CD34⁺ and CD133⁺ hematopoietic stem cells was detected using flow cytometry. CD34⁺ and CD133⁺ cell morphological changes were measured before and after bFGF induction. The concentration and purity of RNA were determined by agarose gel electrophoresis degeneration. Gene-chip test results were analyzed.

RESULTS AND CONCLUSION: ①The 20 samples of cord blood were isolated and purified respectively, CD34⁺ cell purity (77.52±5.06)%, recovery rate (2.74±1.59)%; CD133⁺ cell purity (79.16±3.37) % and the recovery (1.12±0.94)%. ②The new selected CD34⁺ cells were spherical. Following induced by bFGF for 15 days, the majority of cell morphology did not find significant changes, some cells were adherent growth, and protruding and spindle cells were seen. CD133⁺ cells were spherical, by bFGF in cultured 15 days later, cells were significantly amplified, the round shape changed into an irregular shape, and some cells were adherent growth. ③The total RNA of CD34⁺ stem cells before and after incubation was respectively 2 236 ng and 1 796 ng. The total RNA of CD133⁺ stem cells before and after induction was respectively 2 518 ng and 2 191 ng. ④In the detection of 263 genes related to stem cells, two-fold differences of 10 genes in umbilical cord blood CD34⁺ cells and CD133⁺ cells, including five kinds of genes expression were higher in the former than the latter, five kinds of genes expression were lower in the former than the latter. After bFGF-induced culture, 32 kinds of gene expression of CD133⁺ cells were significantly higher than CD34⁺ cells. Among detected 263 genes, no gene was lower than CD34⁺ cells. There were only a few gene expression differences of fresh-separated cord blood CD133⁺ cells and CD34⁺ cells. The response of CD133⁺ cells to bFGF was significantly stronger than CD34⁺ cells, which manifested cell cycle regulation, signal transduction and differentiation, gene expression enhanced.