真核表达BMP4成熟肽及其对iPS细胞的分化抑制作用

Abstract

Abstract: Objective To investigate the role of bone morphogenetic protein4(BMP4) in culturing induced pluripotent stem
cells (iPSCs) and the related signal pathways.MethodsWe amplified the mature peptide of BMP4from the placenta through
RT-PCR, and IgK secretion peptide was ligated to the N-terminal of BMP4 mature peptide. The recombinant plasmid
pPYCAG-IgK-BMP4was transfected into293T cells and screened with puromycin, and the positive clones for expressing
BMP4were verified by cell immunofluorescence and Western blotting. To test the bioactivity of BMP4, iPSCs were cultured in
the medium supplemented with leukemia inhibitory factor (LIF) plus the supernatant containing BMP4, and the cell
phenotype, cell differentiation capacity into lineages of the3germ layers and expression levels of pluripotency-associated
genes were investigated.ResultsSmad1was phosphorylated by BMP4from the culture medium. iPSCs cultured in the
medium supplemented with LIFplus the supernatant containing BMP4for3passages maintained the phenotype of stem cells
with the expression levels of pluripotency-associated genes not affected. These iPSCs also maintained the capacity to
differentiate into cell lineages of the3germ layers.ConclusionBMP4can be efficiently expressed in mammalian cells to
maintain the multipotent differentiation capacity of the iPSCs inin vitroculture.

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