Investigation Into The Functional Intersection Of Different Small Chemical Molecules In Maintaining The Pluripotency Of Mouse Embryonic Stem Cells

The unique feature of Embryonic stem cells(ESCs)—infinite self-renewal and pluripotency, confers ESCs an unparalleled position in fundamental research and regenerative medicine. Hence it is vital to precisely manipulate the fate of ESCs, that is, to sustain pluripotency(self-renewal) or to exert pluripotency(lineage specification). Now it is widely believed that transcriptional regulatory network centered on key transcription factors are the scaffold for maintaining ESCs pluripotency.Small molecules(SMs), small non-peptide, organic compounds that can recognize and bind specific high structure of protein, are usually used in targeted chemotherapy against cancer. Putting SMs into stem cell field produces great success, some research achievements even break the conventional understanding in mechanisms about how ESCs pluripotency is sustained. As a novel research tool, there is still too much to explore in the detailed mechanisms by which SMs maintain pluripotency. During the last decade, multiple SMs have been reported to prompt ESCs pluripotency, however, these studies usually focus on individual small molecule and lay too much emphasis on the individual function of some small molecule. In this way, it is always puzzling to pick up important regulatory effect from extensive influences caused by small molecule in ESCs. That SMs often have pleiotropic effects reportedly make it even harder to determine whether the extensive biological influence caused by SMs are beneficial for maintaining pluripotency. We surmise that by overlapping the altered activities in ESCs caused by different SMs will provide more insights into uncovering more master effects of small molecules as well as to probe the innate pluripotency-related program.For the first time, we horizontally compared the altered biological activities caused by different SMs based on the genome-wide gene expression data, which can be considered an effort to locate where all the small molecules converge on. We used four SMs CHIR99021, PD0325901, SC1 and SB431542 to treat mouse J1 ESCs, and performed a microarray analysis for readouts of SMs. After a multi-angle bioinformatics analysis of microarray data and experimental validation, we can tentatively put forward the following viewpoints:a. SMs CHIR99021、PD0325901、SC1 and SB431542 prompt ESCs pluripotency with a dual-acting mechanism — upregulating pluripotency genes and downregulating differentiation-related genes, which accords with the view that the nature of ESCs pluripotency lies in the balance between pluripotency genes and differentiation-related genes.b. There is an underestimated link between protein component in plasma membrane and extracellular matrix and ESCs pluripotency. The notorious biochemical properties of membrane protein hampered the research progress, which may be a novel breakthrough point in ESCs field when the technical problems in proteomics are solved. And consistent with this view, researchers utilized the biophysical feature of cell culture labware to enhance reprogramming and another group found that cellular mechanical force play a role in reprogramming.c. From a perspective of metabolism, it is essential to maintain a high level of glycolysis and metabolites(for example, linoleic acid) with highly unsaturated for the self-renewal of ESCs, which might be a shared mechanism of multiple SMs in prompting pluripotency. A very recent report implied that it might produce new concepts about maintaining ESCs pluripotency if we purely work on how metabolism establishes pluripotency.d. A direct or indirect repression of MAPK pathway and TGF-beta pathway is a common effect of different SMs, which indicates that the activation of these two signaling pathways are primary forces against pluripotency in ESCs. This finding will be of great value in designing pathway specific drugs in cancer treatment.e. Overlapping the responsive genes to four SMs generated two lists of candidate genes for prompting or repressing pluripotency. ESCAPE analysis showed a strong correlation between pluripotency and most candidate genes. Except for Nanog, Prdm14 and Tcfcp2l1, the rest candidate genes are barely reported in ESCs, so they are likely to be the missing but important genes in pluripotency regulating network.f. We explored the roles of two candidate genes, Lrrc34 and Hesx1, in ESCs pluripotency, and we obtained positive results. Halfway we run more tests on Lrrc34, a publication revealing the relationship between lrrc34 and ESCs pluripotency emerged, which of course supported our view extracted from chip data that the overlapping influence of different SMs accounts.g. By constructing inducible Hesx1 and shRNA targeting Hesx1, we proved that Hesx1 prompts ESCs pluripotency and antagonized the differentiation. The underlying mechanisms might be the elevated expression level of Nanog in Hesx1 overexpressing cells.h. LIF, CHIR99021 and PD0325901 induce Hesx1 independently. In in-vitro culture systems for mouse ESCs, Hesx1 can partially replace LIF, CHIR99021 or PD0325901 to support ESCs self-renewal, which confirms our hypothesis that a gene lied at the intersection of different SMs possesses significant function.Taken together, we invent a novel method to uncover neglected or missing genes in maintaining pluripotency. This study lays both theoretical and experimental foundation for a better understanding of mechanism shared by different SMs in sustaining pluripotency, as well as is insightful for dissecting the multiple dimensions in the maintenance of ESCs pluripotency.