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      Downregulated nuclear lncRNA NRON inhibits SHP2/Wnt/β-catenin signaling and cardiomyocyte differentiation during the development of Tetralogy of Fallot

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          Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling.

          Human pluripotent stem cells (hPSCs) offer the potential to generate large numbers of functional cardiomyocytes from clonal and patient-specific cell sources. Here we show that temporal modulation of Wnt signaling is both essential and sufficient for efficient cardiac induction in hPSCs under defined, growth factor-free conditions. shRNA knockdown of β-catenin during the initial stage of hPSC differentiation fully blocked cardiomyocyte specification, whereas glycogen synthase kinase 3 inhibition at this point enhanced cardiomyocyte generation. Furthermore, sequential treatment of hPSCs with glycogen synthase kinase 3 inhibitors followed by inducible expression of β-catenin shRNA or chemical inhibitors of Wnt signaling produced a high yield of virtually (up to 98%) pure functional human cardiomyocytes from multiple hPSC lines. The robust ability to generate functional cardiomyocytes under defined, growth factor-free conditions solely by genetic or chemically mediated manipulation of a single developmental pathway should facilitate scalable production of cardiac cells suitable for research and regenerative applications.
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            Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex.

            Nuclear factor of activated T cells (NFAT) proteins are Ca(2+)-regulated transcription factors that control gene expression in many cell types. NFAT proteins are heavily phosphorylated and reside in the cytoplasm of resting cells; when cells are stimulated by a rise in intracellular Ca(2+), NFAT proteins are dephosphorylated by the Ca(2+)/calmodulin-dependent phosphatase calcineurin and translocate to the nucleus to activate target gene expression. Here we show that phosphorylated NFAT1 is present in a large cytoplasmic RNA-protein scaffold complex that contains a long intergenic noncoding RNA (lincRNA), NRON [noncoding (RNA) repressor of NFAT]; a scaffold protein, IQ motif containing GTPase activating protein (IQGAP); and three NFAT kinases, casein kinase 1, glycogen synthase kinase 3, and dual specificity tyrosine phosphorylation regulated kinase. Combined knockdown of NRON and IQGAP1 increased NFAT dephosphorylation and nuclear import exclusively after stimulation, without affecting the rate of NFAT rephosphorylation and nuclear export; and both NRON-depleted T cells and T cells from IQGAP1-deficient mice showed increased production of NFAT-dependent cytokines. Our results provide evidence that a complex of lincRNA and protein forms a scaffold for a latent transcription factor and its regulatory kinases, and support an emerging consensus that lincRNAs that bind transcriptional regulators have a similar scaffold function.
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              An episomal vector-based CRISPR/Cas9 system for highly efficient gene knockout in human pluripotent stem cells

              Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.
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                Author and article information

                Contributors
                Journal
                Genes Dis
                Genes Dis
                Genes & Diseases
                Chongqing Medical University
                2352-4820
                2352-3042
                23 October 2022
                May 2023
                23 October 2022
                : 10
                : 3
                : 750-752
                Affiliations
                [a ]Zhang Zhongjing School of Chinese Medicine; Henan Key Laboratory of Zhang Zhongjing Formulae and Herbs for Immunoregulation, Nanyang Institute of Technology, Nanyang, Henan 473004, China
                [b ]Department of Cardiology, Heart Centre; Ministry of Education Key Laboratory of Child Development and Disorders; National Clinical Research Center for Child Health and Disorders; Chongqing Key Laboratory of Pediatrics China International Science and Technology Cooperation Base of Child Development and Critical Disorders; Children's Hospital of Chongqing Medical University, Chongqing 400014, China
                [c ]Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Fudan University, Shanghai 200011, China
                [d ]Obstetrics and Gynecology Hospital, NHC Key Laboratory of Reproduction Regulation, Shanghai Institute of Planned Parenthood Research, State Key Laboratory of Genetic Engineering at School of Life Sciences, Children's Hospital, Fudan University, Shanghai 200438, China
                Author notes
                []Corresponding author. jietian@ 123456cqmu.edu.cn
                [∗∗ ]Corresponding author. Shanghai Key Laboratory of Metabolic Remodeling and Health, Institute of Metabolism and Integrative Biology, Fudan University, Shanghai 200011, China. wanghy@ 123456fudan.edu.cn
                Article
                S2352-3042(22)00279-3
                10.1016/j.gendis.2022.10.010
                10308166
                37396545
                e339e49f-da9e-4dc7-9546-658707a1d1ff
                © 2022 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd.

                This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

                History
                : 2 April 2022
                : 18 July 2022
                : 14 October 2022
                Categories
                Rapid Communication

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