The Function Of SP5 In Regulating Embryonic Stem Cell Self-Renewal

Mouse embryonic stem cells (mESC) are pluripotent cells isolated from the inner cell mass (ICM) of the pre-implantation blastocysts, which has the capacity to self-renewal via propagating indefinitely, and to differentiate in multiple directions. LIF/serum medium was used earliestly to culture mESCs in the absence of feeder cells. Meanwhile Ying et al. found that mESCs pluripotent state can be maintained in serum-free medium N2B27 by addition of two small molecule inhibitors (2i) CHIR and PD03. Both of this two culture conditions can maintain mESCs in pluripotent state by itself pathways, while the researchers found LIF could support mESCs self-renewal in the absence of serum when combined with CHIR or PD03. Meanwhile the research from lyashenko have shown:LIF/PD03 conditions was sufficient to maintain self-renewal in β-catenin-null mESCs, suggesting that LIF and CHIR may maintain self-renewal of stem cells by stimulating the common target genes.In order to screen these genes, mESCs cultured in LIF and LIF/CHIR were used for DNA microarray experiments. As a result,68 genes treated with CHIR stimulus were upregulated more than 1.5-fold. Compared those with STAT3 downstream genes, Sp5 and Trh were considered to be up-regulated simultaneously by Stat3-actived and CHIR, which was confirmed by qRT-PCR. We hypothesize that LIF and CHIR converge on sp5 to promote mESCs self-renewal.Depending on the upregulation of LIF to SP5,46C mESCs overexpressing sp5 were cultured in serum medium without LIF. The results showed that:(1) PB-SP5 cells kept typical morphology of mESCs, alkaline phosphatase staining (AP) was positive, while PB cells died or differentiated, AP staining was negative; (2) qRT-PCR analysis showed that:PB-SP5 cells had high expression of genes related to pluripotency, and lower expression of differentiation-related genes; (3) immunofluorescence staining of Oct4, Nanog, Klf4, etc. was positive; (4) Cells maintained the ability to normally differentiated into three germ-layers in the excision of SP5. These results indicate that overexpression of SP5 can relieve dependence of mESCs for LIF. At the same time, we found that:(1) LIF was failed to increase the expression of SP5 in presence of inhibitor of LIF/Stat3; (2) SP5 had a high expression in Stat3-activated cells; (3) SP5 overexpression can maintain the ability of self-renewal in Stat3-null mESCs. These results show that:LIF can upregulate the expression of SP5 to support self-renewal in mESCs via LIF/Stat3 signaling pathway. But it is worth to note that knockdown of SP5 could not damage 46C mESCs self-renewal, suggesting SP5 may be just one of the downstream target genes promoting self-renewal.Furtherly, we verify whether CHIR promote mESCs self-renewal by upregulating the expression of SP5. The results showed that:(1) SP5 could partically relieve dependence of mESCs for LIF; (2) The extension of CHIR stimulation could increase the expression of SP5; (3) But CHIR is failed to upregulate expression of SP5 in β-catenin-null mESCs. Therefore, SP5 can partially replace the role of CHIR in 2i maintaining self-renewal.Mesodermal embryonic stem cells (EpiSCs) are isolated from epiblasts of post-implantation embryos, some of LIF/Stat3 target genes could initiate EpiSCs to recode into ESCs state. The expression of SP5 had no significant difference between mESCs and EpiSCs via qRT-PCR examination, however, some ESCs-like clones had shown in EpiSCs over-expressing SP5 when cultured in LIF/Serum/2i, and could continually expand in LIF/serum, and expressed ESs marker genes. Meanwhile, OCRG9 EpiSCs possessing Oct4-GFP reporter gene could emit green fluorescence and express marker genes through the similar processing. These results illustrate SP5 can promote EpiSCs recoding into ES state.In conclusion, both of LIF/Stat3 pathways and Wnt/β-catenin pathways could upregulate the expression sp5 which has the capacity to recode EpiSCs to ESCs to promote mESCs self-renewal.As a new member of regulatory networks of self-renewal-promoting, discovery of SP5 will deepen our understanding of the mechanisms.