Background: The relationship between somatic cell proliferation and reprogramming efficiency remains elusive.Results: Reducing somatic cell proliferation by removing c-Myc or adding anti-proliferative compounds dramatically improves induced pluripotent stem cell generation.Conclusion: Hyperproliferation of somatic cells might be detrimental to reprogramming.Significance: The current study provides new perspectives upon the connection between somatic cell proliferation control and reprogramming.
G protein-coupled receptor 3 (GPR3) is an orphan G protein-coupled receptor (GPCR) predominantly expressed in mammalian brain and oocytes. GPR3 plays important roles in these two organs and is known as a Gαs-coupled receptor-activated constitutively in cells. However, the signal transduction pathway and pharmacological function of GPR3 remain unclear because of the lack of a specific ligand. By use of a human embryonic kidney 293 cell line stably expressing FLAG-GPR3-green fluorescent protein, a chemical screening for GPR3 ligands was performed using homogeneous time-resolved fluorescence cAMP assay. Diphenyleneiodonium chloride (DPI) was identified as a novel agonist of GPR3 with weak or no cross-reactivity with other GPCRs. DPI was further characterized to activate several GPR3-mediated signal transduction pathways, including Ca(2+) mobilization, cAMP accumulation, membrane recruitment of β-arrestin2, and receptor desensitization. Parallel studies revealed that the activity of DPI is much more pronounced than sphingosine 1-phosphate, a previously reported GPR3 agonist. Our study identified a novel and specific agonist of GPR3, which provides a useful tool for further study of this orphan GPCR.
The revolutionary induced pluripotent stem cell (iPSC) technology provides a new means for cell replacement therapies and drug screening. Small molecule compounds have been found extremely useful to improve the generation of iPSCs and understand the reprogramming mechanism. Here we report the identification of a novel chemical, CYT296, which improves OSKM-mediated induction of iPSCs for >10 folds and enables efficient reprogramming with only Oct4 in combination with other small molecules. The derived iPSCs are genuinely pluripotent and support the development of two 'All-iPSC' mice by tetraploid complementation. CYT296 profoundly impacts heterochromatin formation without affecting cell viability. MEFs treated with CYT296 exhibit de-condensed chromatin structure with markedly reduced loci containing heterochromatin protein 1α (HP1α) and H3K9me3, which is very similar to the chromatin configuration in embryonic stem cells (ESCs). Given that an open chromatin structure serves as a hallmark of pluripotency and has to be acquired to fulfill reprogramming, we propose that CYT296 might facilitate this process by disrupting condensed chromatin, thereby creating a more favorable environment for reprogramming. In agreement of this idea, shRNA targeting HP1α also promotes the generation of iPSCs. Thus current findings not only provide a novel chemical for efficient iPSC induction, but also suggest a new approach to regulate somatic cell reprogramming by targeting chromatin de-condensation with small molecules.
G protein-coupled receptors (GPCRs) are important modulators of many physiological functions and excellent drug targets for many diseases. However, to study the functions of endogenous GPCRs is still a challenging task, partially due to the low expression level of GPCRs and the lack of highly potent and selective GPCR antibodies. Overexpression or knock-in of tagged GPCRs, or knockout of specific GPCRs in mice, are common strategies used to study the in vivo functions of these receptors. However, generating separate mice carrying tagged GPCRs or conditional alleles for GPCRs is labor intensive, and requires additional breeding costs. Here we report the generation of mice carrying an HA-tagged DOR (delta opioid receptor) flanked by LoxP sequences at the endogenous DOR locus using a single recombination step, aided by the TALEN system. These animals can be used directly to study the expression, localization, protein-protein interaction and signal transduction of endogenous DOR using anti-HA antibodies. By crossing with mice expressing tissue-specific Cre, these mice can also generate offspring with DOR knockout within specific tissues. These mice are powerful tools to study the in vivo functions of DOR. Furthermore, the gene modification strategy could also be used to study the functions of many other GPCRs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.