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. Through transcriptome and methylome analysis, we discovered that the epigenetic repression of CHCHD2, a mitochondrial protein, is a common occurrence during in vitro culture using enzymatic dissociation. We confirmed this finding through genetic perturbation and reconstitution experiments in normal human embryonic stem cells (hESCs). Loss of CHCHD2 expression conferred resistance to single cell dissociation-induced cell death, a common stress encountered during in vitro culture. Importantly, we found that the downregulation of CHCHD2 significantly attenuates the activity of Rho-associated protein kinase (ROCK), which is responsible for inducing single cell death in hESCs. This suggests that hESCs may survive routine enzyme-based cell dissociation by downregulating CHCHD2 and thereby attenuating ROCK activity. These findings provide insights into the mechanisms by which hPSCs acquire survival advantages and adapt to in vitro culture conditions.

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. Through high-resolution genome-wide approaches and transcriptome analysis, we found that culture adapted-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2, a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stabilization, misaligned chromosomes, and polyploidy. CONCLUSION: These studies suggest that the increased transcription of TPX2 in culture adapted hESCs could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

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. However, the underlying mechanisms for BCL2L1 induction remain unknown. Furthermore, abnormal mitosis and the survival advantage that frequently occur in late passage are associated with the expression of BCL2L1, which is in locus 20q11.21. In this study, we demonstrated that the expression of TPX2, a gene located in 20q11.21, led to BCL2L1 induction and consequent survival traits under mitotic stress in isogenic pairs of hESCs and human induced pluripotent stem cells (iPSCs) with normal and 20q11.21 CNVs. High Aurora A kinase activity by TPX2 stabilized the YAP1 protein to induce YAP1-dependent BCL2L1 expression. A chemical inhibitor of Aurora A kinase and knockdown of YAP/TAZ significantly abrogated the high tolerance to mitotic stress through BCL2L1 suppression. These results suggest that the collective expression of TPX2 and BCL2L1 from CNV at loci 20q11.21 and a consequent increase in YAP1 signaling promote genome instability during long-term in vitro hESC culture.

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. This study reports an electrochemical method that enables simple, label-free, and non-destructive detection of naïve embryonic stem cells (ESCs) derived from mouse ESCs, based on the differences in cellular metabolism. Two major metabolic pathways to generate adenosine triphosphate (ATP)-glycolysis and oxidative phosphorylation (OXPHOS)-were blocked, and it was found that mitochondrial energy generation is the origin of the strong electrochemical signals of naïve ESCs. The number of ESCs is quantified when mixed with primed ESCs or converted from naïve-primed switchable metastable ESCs. The mouse PSCs derived from doxycycline-inducible mouse embryonic fibroblasts (MEFs) are also sensitively identified among other cell types such as unconverted MEFs and primed PSCs. The developed sensing platform operates in a non-invasive and label-free manner. Thus, it can be useful in the development of stem cell-derived therapeutics.

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. RESULTS: Loss of Shp2 increased naïve 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 iShp2 can replace the use of iMek1 for maintenance of naïve ESCs. CONCLUSIONS: 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.

L1 retrotransposons exploit RNA m6A modification as an evolutionary driving force.

L1 retrotransposons can pose a threat to genome integrity. The host has evolved to restrict L1 replication. However, mechanisms underlying L1 propagation out of the host surveillance remains unclear. Here, we propose an evolutionary survival strategy of L1, which exploits RNA m6A modification. We discover that m6A 'writer' METTL3 facilitates L1 retrotransposition, whereas m6A 'eraser' ALKBH5 suppresses it. The essential m6A cluster that is located on L1 5' UTR serves as a docking site for eukaryotic initiation factor 3 (eIF3), enhances translational efficiency and promotes the formation of L1 ribonucleoprotein. Furthermore, through the comparative analysis of human- and primate-specific L1 lineages, we find that the most functional m6A motif-containing L1s have been positively selected and became a distinctive feature of evolutionarily young L1s. Thus, our findings demonstrate that L1 retrotransposons hijack the RNA m6A modification system for their successful replication.

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. The two unique orthogonal mechanisms provide the potential of multi-demensional cellular distinction of specific cell types.

Luteolin Induces Selective Cell Death of Human Pluripotent Stem Cells.

Despite recent advances in clinical stem cell therapy applications based on human pluripotent stem cells (hPSCs), potential teratoma formation due to the presence of residual undifferentiated hPSCs remains a serious risk factor that challenges widespread clinical application. To overcome this risk, a variety of approaches have been developed to eliminate the remaining undifferentiated hPSCs via selective cell death induction. Our study seeks to identify natural flavonoids that are more potent than quercetin (QC), to selectively induce hPSC death. Upon screening in-house flavonoids, luteolin (LUT) is found to be more potent than QC to eliminate hPSCs in a p53-dependent manner, but not hPSC-derived smooth muscle cells or perivascular progenitor cells. Particularly, treating human embryonic stem cell (hESC)-derived cardiomyocytes with LUT efficiently eliminates the residual hESCs and only results in marginal effects on cardiomyocyte (CM) functions, as determined by calcium influx. Considering the technical limitations of isolating CMs due to a lack of exclusive surface markers at the end of differentiation, LUT treatment is a promising approach to minimize teratoma formation risk.

Safe scarless cassette-free selection of genome-edited human pluripotent stem cells using temporary drug resistance.

An efficient gene-editing technique for use in human pluripotent stem cells (hPSCs) has great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the extremely low efficiency of gene editing in hPSCs remains as a major technical hurdle. Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to the high expression of SLC35F2 in these cells. Knockout of SLC35F2 with CRISPR-Cas9, or depletion with siRNAs, made the hPSCs highly resistant to YM155. Simultaneous editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled the survival of genome-edited hPSCs as a result of temporary YM155 resistance, thereby achieving an enriched selection of clonal populations with gene knockout or knock-in. This precise and efficient genome editing approach took as little as 3 weeks and required no cell sorting or the introduction of additional genes, to be a more feasible approach for gene editing in hPSCs due to its simplicity.

Structure-Activity Relationship Analysis of YM155 for Inducing Selective Cell Death of Human Pluripotent Stem Cells.

Despite great potential for regenerative medicine, the high tumorigenic potential of human pluripotent stem cells (hPSCs) to form undesirable teratoma is an important technical hurdle preventing safe cell therapy. Various small molecules that induce the complete elimination of undifferentiated hPSCs, referred to as "stemotoxics," have been developed to facilitate tumor-free cell therapy, including the Survivin inhibitor YM155. In the present work, based on the chemical structure of YM155, total 26 analogs were synthesized and tested for stemotoxic activity toward human embryonic stem cells (hESCs) and induced PSCs (iPSCs). We found that a hydrogen bond acceptor in the pyrazine ring of YM155 derivatives is critical for stemotoxic activity, which is completely lost in hESCs lacking SLC35F2, which encodes a solute carrier protein. These results suggest that hydrogen bonding interactions between the nitrogens of the pyrazine ring and the SLC35F2 protein are critical for entry of YM155 into hPSCs, and hence stemotoxic activity.

A Theoretical Model for the Cell Cycle and Drug Induced Cell Cycle Arrest of FUCCI Systems with Cell-to-Cell Variation during Mitosis.

PURPOSE: Since the molecular mechanism of the cell cycle was established, various theoretical models of this process have been developed. A recent study revealed significant variability in cell cycle duration between mother and daughter cells, but this observation has not been incorporated into the theoretical models. METHODS: We used fluorescent ubiquitination-based cell cycle indicator (FUCCI) systems and live-monitored the heterogeneity of cell cycle progression within daughter cells, which accounts for dephasing synchrony. To incorporate the variable cell cycle durations into a model, we modified a two-ordinary differential equation (ODE) model based on reciprocal activation between CDK1 and APC. RESULTS: Our model reproduced the experimental population profile, in which cell cycle synchrony dephased due to variability. Based on this model, we determined parameters for CDK1 and APC in the cell cycle profile after treatment with antimitotic drugs and associated the parameters with the drugs' mode of action as cell cycle inhibitors. CONCLUSION: This suggests that this model is useful for determining the mode of action of unknown small molecules on the cell cycle.

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

Human and mouse embryonic stem cells (ESCs) differ in terms of their pluripotency status, i.e., naïve vs. primed. This affects various biological properties and leads to several technical hurdles for future clinical applications, such as difficulties in chimera formation, single-cell passaging, and gene editing. In terms of generating functional human tissues and organs via mammalian interspecies chimerism, a fluorescent chemical probe that specifically labels naïve ESCs would help to isolate these cells and monitor their conversion. This study demonstrates that the fluorescent chemical probe compound of designation yellow 9 (CDy9) selectively stains naïve, but not primed, mouse ESCs (mESCs). CDy9 entered cells via Slc13a5, a highly expressed membrane transporter in naïve mESCs. Fluorescence-based cell sorting based on CDy9 staining successfully separated naïve mESCs from primed mESCs. Mice generated using CDy9+ cells isolated during the conversion of mouse epiblast stem cells into naïve mESCs exhibited coat color chimerism. Furthermore, CDy9 specifically stained cells in the inner cell mass of mouse embryos. These findings suggest that CDy9 is a useful tool to isolate functional naïve mESCs.

Screening of cytotoxic or cytostatic flavonoids with quantitative Fluorescent Ubiquitination-based Cell Cycle Indicator-based cell cycle assay.

The Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) system can be used not only to study gene expression at a specific cell cycle stage, but also to monitor cell cycle transitions in real time. In this study, we used a single clone of FUCCI-expressing HeLa cells (FUCCI-HeLa cells) and monitored the cell cycle in individual live cells over time by determining the ratios between red fluorescence (RF) of RFP-Cdt1 and green fluorescence (GF) of GFP-Geminin. Cytotoxic and cytostatic compounds, the latter of which induced G2 or mitotic arrest, were identified based on periodic cycling of the RF/GF and GF/RF ratios in FUCCI-HeLa cells treated with anti-cancer drugs. With this cell cycle monitoring system, ten flavonoids were screened. Of these, apigenin and luteolin, which have a flavone backbone, were cytotoxic, whereas kaempferol, which has a flavonol backbone, was cytostatic and induced G2 arrest. In summary, we developed a system to quantitatively monitor the cell cycle in real time. This system can be used to identify novel compounds that modulate the cell cycle and to investigate structure-activity relationships.

Role of MEK partner-1 in cancer stemness through MEK/ERK pathway in cancerous neural stem cells, expressing EGFRviii.

BACKGROUND: Glioma stem cells (GSCs) are a major cause of the frequent relapse observed in glioma, due to their high drug resistance and their differentiation potential. Therefore, understanding the molecular mechanisms governing the 'cancer stemness' of GSCs will be particularly important for improving the prognosis of glioma patients. METHODS: We previously established cancerous neural stem cells (CNSCs) from immortalized human neural stem cells (F3 cells), using the H-Ras oncogene. In this study, we utilized the EGFRviii mutation, which frequently occurs in brain cancers, to establish another CNSC line (F3.EGFRviii), and characterized its stemness under spheroid culture. RESULTS: The F3.EGFRviii cell line was highly tumorigenic in vitro and showed high ERK1/2 activity as well as expression of a variety of genes associated with cancer stemness, such as SOX2 and NANOG, under spheroid culture conditions. Through meta-analysis, PCR super-array, and subsequent biochemical assays, the induction of MEK partner-1 (MP1, encoded by the LAMTOR3 gene) was shown to play an important role in maintaining ERK1/2 activity during the acquisition of cancer stemness under spheroid culture conditions. High expression of this gene was also closely associated with poor prognosis in brain cancer. CONCLUSION: These data suggest that MP1 contributes to cancer stemness in EGFRviii-expressing glioma cells by driving ERK activity.

BCL2 induced by LAMTOR3/MAPK is a druggable target of chemoradioresistance in mesenchymal lung cancer.

Mesenchymal-type cancers after epithelial mesenchymal transition (EMT) were recently shown to acquire chemoresistance through expressing EMT specific transcription factors. However, druggable (or actionable) target(s) for chemoresistance in mesenchymal-type lung cancers remain unidentified. Here, we used a public clinical genomic database and mesenchymal lung cancer cells (MLCC) model derived from the A549 lung adenocarcinoma cell line to demonstrate that BCL2 expression, which is highly induced in mesenchymal-type lung cancers, as a predictor of poor prognosis in mesenchymal lung cancer patients and association with acquired chemoradioresistance. Thereby, combination treatment with BH3 mimetics, such as ABT-263 and ABT-737, clearly attenuated chemoresistance in MLCCs. BCL2 expression in MLCCs was induced by ERK1 activity through the upregulation of the MEK1/ERK1 scaffold protein MEK partner-1 (MP1). Interfering with the MEK1/MP1/ERK1 axis using a MEK1 inhibitor or MP1 depletion repressed BCL2 expression and sensitized MLCCs to chemoradiotherapy. Taken together, our results suggest that targeting druggable proteins in the MEK1/MP1/ERK1/BCL2 axis, such as MEK1 or BCL2, with currently available FDA approved drugs is a currently feasible approach to improve clinical outcomes of mesenchymal lung cancer patients.

PRMT8 Controls the Pluripotency and Mesodermal Fate of Human Embryonic Stem Cells By Enhancing the PI3K/AKT/SOX2 Axis.

Basic fibroblast growth factor (bFGF) supplementation is critical to maintain the pluripotency of human pluripotent stem cells (hPSCs) through activation of PI3K/AKT, rather than MEK/ERK pathway. Thus, elaborate molecular mechanisms that preserve PI3K/AKT signaling upon bFGF stimulation may exist in hPSCs. Protein arginine methyltransferase 8 (PRMT8) was expressed and then its level gradually decreased during spontaneous differentiation of human embryonic stem cells (hESCs). PRMT8 loss- or gain-of-function studies demonstrated that PRMT8 contributed to longer maintenance of hESC pluripotency, even under bFGF-deprived conditions. Direct interaction of membrane-localized PRMT8 with p85, a regulatory subunit of PI3K, was associated with accumulation of phosphoinositol 3-phosphate and consequently high AKT activity. Furthermore, the SOX2 induction, which was controlled by the PRMT8/PI3K/AKT axis, was linked to mesodermal lineage differentiation. Thus, we propose that PRMT8 in hESCs plays an important role not only in maintaining pluripotency but also in controlling mesodermal differentiation through bFGF signaling toward the PI3K/AKT/SOX2 axis. Stem Cells 2017;35:2037-2049.