Mammalian ubiquitous promoter isolated from proximal regulatory region of bovine MSTN gene.

Gene therapy is a promising method for treating inherited diseases by directly delivering the correct genetic material into patient cells. However, the limited packaging capacity of vectors poses a challenge. Minimizing promoter size is a viable strategy among various approaches to address this issue.

Longitudinal analysis of genetic and epigenetic changes in human pluripotent stem cells in the landscape of culture-induced abnormality.

Human embryonic stem cells (hESCs) are naturally equipped to maintain genome integrity to minimize genetic mutations during early embryo development. However, genetic aberration risks and subsequent cellular changes in hESCs during in vitro culture pose a significant threat to stem cell therapy. While a few studies have reported specific somatic mutations and copy number variations (CNVs), the molecular mechanisms underlying the acquisition of 'culture-adapted phenotypes' by hESCs are largely unknown.

Remodeling of sorafenib as an orally bioavailable ferroptosis inducer for Lung Cancer by chemical modification of adenine-binding motif.

Sorafenib (BAY 43-9006) was developed as a multi-kinase inhibitor to treat advanced renal cell, hepatocellular, and thyroid cancers. The cytotoxic effect of sorafenib on cancer cells results from not only inhibiting the MEK/ERK signaling pathway (the on-target effect) but also inducing oxidative damage (the off-target effect). The inhibitory effect of sorafenib on system Xc (xCT), a cystine/glutamate antiporter, promotes ferroptosis induction and accounts for oxidative damage.

Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways.

Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived hPSCs. Unlike Cas9-mediated knock-in, cytosine base editor and prime editor achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death.

Systematic omics analysis identifies CCR6 as a therapeutic target to overcome cancer resistance to EGFR inhibitors.

Epidermal growth factor receptor inhibitors (EGFRi) have exhibited promising clinical outcomes in the treatment of various cancers. However, their widespread application has been limited by low patient eligibility and the emergence of resistance. Leveraging a multi-omics approach (>1000 cancer cell lines), we explored molecular signatures linked to EGFRi responsiveness and found that expression signatures involved in the estrogen response could recapitulate cancer cell dependency on EGFR, a phenomenon not solely attributable to EGFR-activating mutations.

Crosstalk between Signaling Pathways and Energy Metabolism in Pluripotency.

The sequential change from totipotency to multipotency occurs during early mammalian embryo development. However, due to the lack of cellular models to recapitulate the distinct potency of stem cells at each stage, their molecular and cellular characteristics remain ambiguous. The establishment of isogenic naïve and primed pluripotent stem cells to represent the pluripotency in the inner cell mass of the pre-implantation blastocyst and in the epiblast from the post-implantation embryo allows the understanding of the distinctive characteristics of two different states of pluripotent stem cells.

Epigenetic repression of CHCHD2 enhances survival from single cell dissociation through attenuated Rho A kinase activity.

During in vitro culture, human pluripotent stem cells (hPSCs) often acquire survival advantages characterized by decreased susceptibility to mitochondrial cell death, known as "culture adaptation." This adaptation is associated with genetic and epigenetic abnormalities, including TP53 mutations, copy number variations, trisomy, and methylation changes. Understanding the molecular mechanisms underlying this acquired survival advantage is crucial for safe hPSC-based cell therapies.

Partial in vivo reprogramming enables injury-free intestinal regeneration via autonomous induction.

Tissue regeneration after injury involves the dedifferentiation of somatic cells, a natural adaptive reprogramming that leads to the emergence of injury-responsive cells with fetal-like characteristics. However, there is no direct evidence that adaptive reprogramming involves a shared molecular mechanism with direct cellular reprogramming. Here, we induced dedifferentiation of intestinal epithelial cells using OSKM (Oct4, Sox2, Klf4, and c-Myc) in vivo.

In Silico Discovery of 5'-Modified 7-Deoxy-7-ethynyl-4'-thioadenosine as a HASPIN Inhibitor and Its Synergistic Anticancer Effect with the PLK1 Inhibitor.

Despite genetic perturbations resulting in embryo lethality for most mitotic kinases, loss of the histone H3 mitotic kinase HASPIN reveals no adverse effect in mice models, establishing HASPIN as a promising target for anticancer therapy. However, developing a HASPIN inhibitor from conventional pharmacophores poses a technical challenge as this atypical kinase shares slight similarities with eukaryotic protein kinases. Chemically modifying a cytotoxic 4'-thioadenosine analogue through high genotoxicity yielded several novel nongenotoxic kinase inhibitors.

MutSα and MutSβ as size-dependent cellular determinants for prime editing in human embryonic stem cells.

Precise genome editing in human pluripotent stem cells (hPSCs) has potential applications in isogenic disease modeling and stem cell therapy, necessitating diverse genome editing tools. However, unlike differentiated somatic cells, hPSCs have unique cellular properties that maintain genome integrity, which largely determine the overall efficiency of an editing tool. Considering the high demand for prime editors (PEs), it is imperative to characterize the key molecular determinants of PE outcomes in hPSCs.

Gene editing with 'pencil' rather than 'scissors' in human pluripotent stem cells.

Owing to the advances in genome editing technologies, research on human pluripotent stem cells (hPSCs) have recently undergone breakthroughs that enable precise alteration of desired nucleotide bases in hPSCs for the creation of isogenic disease models or for autologous ex vivo cell therapy. As pathogenic variants largely consist of point mutations, precise substitution of mutated bases in hPSCs allows researchers study disease mechanisms with "disease-in-a-dish" and provide functionally repaired cells to patients for cell therapy. To this end, in addition to utilizing the conventional homologous directed repair system in the knock-in strategy based on endonuclease activity of Cas9 (i.

TPX2 Amplification-Driven Aberrant Mitosis in Culture Adapted Human Embryonic Stem Cells with gain of 20q11.21.

Background: Despite highly effective machinery for the maintenance of genome integrity in human embryonic stem cells (hESCs), the frequency of genetic aberrations during in-vitro culture has been a serious issue for future clinical applications.

Method: By passaging hESCs over a broad range of timepoints (up to 6 years), the isogenic hESC lines with different passage numbers with distinct cellular characteristics, were established.

Result: We found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs) with normal copy number.

Transition Substitution of Desired Bases in Human Pluripotent Stem Cells with Base Editors: A Step-by-Step Guide.

The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation.

TPX2 prompts mitotic survival via the induction of BCL2L1 through YAP1 protein stabilization in human embryonic stem cells.

Genetic alterations have been reported for decades in most human embryonic stem cells (hESCs). Survival advantage, a typical trait acquired during long-term in vitro culture, results from the induction of BCL2L1 upon frequent copy number variation (CNV) at locus 20q11.21 and is one of the strongest candidates associated with genetic alterations that occur via escape from mitotic stress.

Label-free and non-destructive identification of naïve and primed embryonic stem cells based on differences in cellular metabolism.

Pluripotent stem cells (PSCs) exist in naïve or primed states based on their origin. For in vitro culture, these PSCs require different supplements and growth factors. However, owing to their similar phenotypic features, identifying both cell types without harming cellular functions is challenging.

Activation of the HSP27-AKT axis contributes to gefitinib resistance in non-small cell lung cancer cells independent of EGFR mutations.

Purpose: Although epidermal growth factor receptor (EGFR)-activating mutations in non-small cell lung cancer (NSCLC) usually show sensitivity to first-generation EGFR-tyrosine kinase inhibitors (TKIs), most patients relapse because of drug resistance. Heat shock protein 27 (HSP27) has been reported to be involved in the resistance of EGFR-TKIs, although the underlying mechanism is unclear. Here, we explore the mechanisms of HSP27-mediated EGFR TKI resistance and propose novel therapeutic strategies.

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

Background: 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.

Methods: 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.

Live isolation of naïve ESCs via distinct glucose metabolism and stored glycogen.

Naïve and primed pluripotent stem cells recapitulate the peri- and post-implantation development, respectively. Thus, investigation of distinct traits between each pluripotent stem cell type would shed light on early embryonic processes. Herein, by screening a fluorescent probe library, we found that intracellular glycogen led to specific reactivity to CDg4, a glycogen fluorescence sensor, in both human and mouse naïve embryonic stem cells (ESCs).

Target identification of mouse stem cell probe CDy1 as ALDH2 and Abcb1b through live-cell affinity-matrix and ABC CRISPRa library.

CDy1 is a powerful tool to distingusih embryonic stem cells for reprogramming studies and regeneration medicine. However, the stem cell selectivity mechanism of CDy1 has not been fully understood. Here, we report ALDH2 and ABCB1 as the molecular targets of CDy1, elucidated by live-cell affinity-matrix and ABC transporter CRISPRa library screening.

High expression of uracil DNA glycosylase determines C to T substitution in human pluripotent stem cells.

Precise genome editing of human pluripotent stem cells (hPSCs) is crucial not only for basic science but also for biomedical applications such as stem cell therapy and genetic disease modeling. However, hPSCs have unique cellular properties compared to somatic cells. For instance, hPSCs are extremely susceptible to DNA damage, and therefore Cas9-mediated DNA double-strand breaks (DSB) induce p53-dependent cell death, resulting in low Cas9 editing efficiency.