Expression of BMP4 mature peptide in eukaryotic cells and its differentiation-inhibiting effect in culturing induced pluripotent stem cells.
OBJECTIVE
To investigate the role of bone morphogenetic protein 4 (BMP4) in culturing induced pluripotent stem cells (iPSCs) and the related signal pathways.


METHODS
We amplified the mature peptide of BMP4 from the placenta through RT-PCR, and IgK secretion peptide was ligated to the N-terminal of BMP4 mature peptide. The recombinant plasmid pPYCAG-IgK-BMP4 was transfected into 293T cells and screened with puromycin, and the positive clones for expressing BMP4 were 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 the 3 germ layers and expression levels of pluripotency-associated genes were investigated.


RESULTS
Smad1 was phosphorylated by BMP4 from the culture medium. iPSCs cultured in the medium supplemented with LIF plus the supernatant containing BMP4 for 3 passages 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 the 3 germ layers.


CONCLUSIONS
BMP4 can be efficiently expressed in mammalian cells to maintain the multipotent differentiation capacity of the iPSCs in in vitro culture.
Shanghai University of Traditional Chinese Medicine, Shanghai, China.
Daofang
D
Ding
Zhen
Z
Wang
Hao
H
Xu
Leqin
L
Xu
Qianqian
Q
Liang
Yongjian
Y
Zhao
Qi
Q
Shi
Yongjun
Y
Wang
D000818:Animals; D055415:Bone Morphogenetic Protein 4 / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics*; D002478:Cells, Cultured; D005260:Female; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007145:Immunoglobulin kappa-Chains / Q000235:genetics; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D051379:Mice
C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D007145:Immunoglobulin kappa-Chains