A fluorescent chemical probe CDy9 selectively stains and enables the isolation of live naïve mouse embryonic stem cells

Kwon

et al. 2021

Preprint

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The requirement of the Mek1 inhibitor (iMek1) during naïve pluripotency maintenance results from the activation of the Mek1-Erk1/2 (Mek/Erk) signaling pathway upon leukemia inhibitory factor (LIF) stimulation. Through a meta-analysis of previous genome-wide screening for negative regulators of naïve pluripotency, Ptpn11 (encoding the Shp2 protein, which serves both as a tyrosine phosphatase and putative adapter), was predicted as one of the key factors for the negative modulation of naïve pluripotency through LIF-dependent Jak/Stat3 signaling. Using an isogenic pair of naïve and primed mouse embryonic stem cells (mESCs), we demonstrated the differential role of Shp2 in naïve and primed pluripotency. Loss of Shp2 increased naive pluripotency by promoting Jak/Stat3 signaling and disturbed in vivo differentiation potential. In sharp contrast, Shp2 depletion significantly impeded the self-renewal of ESCs under primed culture conditions, which was concurrent with a reduction in Mek/Erk signaling. Similarly, upon treatment with an allosteric Shp2 inhibitor (iShp2), the cells sustained Stat3 phosphorylation and decoupled Mek/Erk signaling, thus replacing the use of iMek1 not only for maintenance but also for the establishment of naïve ESCs through reprogramming. Taken together, our findings highlight the differential roles of Shp2 in naïve and primed pluripotency and propose the usage of iShp2 instead of iMek1 for the efficient maintenance and establishment of naïve pluripotency.

Section: Role Of Shp2 In Naïve Pluripotencysupporting

confidence: 65%

Dichotomous role of Shp2 for naïve and primed pluripotency maintenance in embryonic stem cells

Kwon

et al. 2021

Preprint

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“…49 Although both stem cell states are pluripotent, naive mESCs have unique properties such as dome-shaped morphology. 50 Characterization and isolation of naive mESCs are essential for embryology. CDy9 was previously developed as a mESC-selective probe by DOFLA.…”

Section: Dofla and Gold Probes Through Slc Transportersmentioning

confidence: 92%

“…51 After CDy9 staining of naive and primed mESCs, the CDy9 fluorescence intensity was much higher in naive mESCs. 50 CDy9 can stain specifically the inner cell mass (ICM) cells, which include genuine naive stem cells, among cells of live mouse blastocysts (Figure 5a). 50 Naive mESCs (CDy9-hi population) can be isolated in the mixture of naive mESCs and primed mESCs with the assistance of FACS (Figure 5b).…”

Section: Dofla and Gold Probes Through Slc Transportersmentioning

confidence: 99%

“…50 CDy9 can stain specifically the inner cell mass (ICM) cells, which include genuine naive stem cells, among cells of live mouse blastocysts (Figure 5a). 50 Naive mESCs (CDy9-hi population) can be isolated in the mixture of naive mESCs and primed mESCs with the assistance of FACS (Figure 5b). 50 Localization of CDy9 is in the cytoplasm in naive mESCs.…”

Section: Dofla and Gold Probes Through Slc Transportersmentioning

confidence: 99%

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Holding-Oriented versus Gating-Oriented Live-Cell Distinction: Highlighting the Role of Transporters in Cell Imaging Probe Development

2019

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Conspectus Small molecule imaging probes are powerful tools to understand complex biological systems. The mainstreams of imaging probe developments have been focused on the target holding of the probes; the holding targets are often cell-type-specific biomarkers. This type of the probe mechanism can be designated as holding-oriented live-cell distinction (HOLD). Our group has worked on the development of cell-type-selective probes using a diversity-oriented fluorescence library approach (DOFLA), where unbiased phenotypic screening is employed using fluorescent library compounds. Through the conventional target identification methods such as an affinity-based analysis, we elucidated that some of the probe mechanisms are HOLD. However, we also realized that sometimes there is no specific holding target for probes or the holding targets are ubiquitous. The observation led us to test an alternative mechanism of cell-type-specific probes as gating-oriented live-cell distinction (GOLD). We started to examine the gating mechanism of probes, which is mainly based on transporters but which does not necessarily require probe holding to cellular targets. Transporters can control the in and out movement of various nutrients and chemicals. Different expression levels of transporters in various cell types could provide the molecular mechanism of differential staining of cells by regulating the intracellular accumulation of a certain specific probe. A number of GOLD probes have been developed by modifying or mimicking endogenous substrates of transporters such as inorganic ions, glucose, amino acids, or neurotransmitters, utilizing broad substrate specificity of transporters. The radiolabeled or fluorophore-conjugated substrate mimetics have been widely used for live cell distinction and various applications such as disease-related cell or tissue imaging. In humans, there are about 400 solute carrier (SLC) transporters and 50 ATP-binding cassette (ABC) transporters. Since some transporters have broad substrate specificity, they can transport not only derivatives of endogenous natural substrates but also totally synthetic diverse imaging probes, such as DOFLA probes. Without preconsidering the structure of endogenous substrates, we recently demonstrated a series of live-cell imaging probes and elucidated their molecular mechanism as a gating one, either by SLC or ABC transporters. Transporter inhibitor panel and CRISPR-based transporter libraries could provide a systematic gating target elucidation platform. Considering the generality of DOFLA and the CRISPR-based genomic tool for transporter systems (>450 in humans), the GOLD approach will offer new insight and promise for unprecedented levels of novel cell imaging probe development.

“…Later, Cho et al found that CDy9 exhibited higher selectivity towards naı ¨ve mESC than primed ESCs, elucidating CDy9 as a naı ¨ve mESC probe. 39 Different types of naı ¨ve mESCs were successfully separated from primed mESCs with CDy9. The gating target of CDy9 was identified as Slc13a5, a highly expressed membrane transporter in naı ¨ve mESCs.…”

Section: Slc Transportersmentioning

confidence: 99%

Fluorescent probe strategy for live cell distinction

Liu

Chang

2022

Chem. Soc. Rev.

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