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  • IGF and PI K signaling in regenerating muscle was tightly


    IGF and PI3K signaling in regenerating muscle was tightly regulated across multiple levels and temporally, especially during the transition between myoblast proliferation and differentiation. In the early period, IGF1 was upregulated and quickly followed by increases in expression and chromatin remodeling of myogenic negative regulators (Mg53, Hdacs, Xbp1, Usf1) that alter how myogenic regulatory factors bind to lineage-specific genes. Starting at 72 hr, IGF1 and negative regulators began to decrease in expression while positive differentiation genes increased in expression, which is consistent with previous observations (Braun and Gautel, 2011; Buckingham and Rigby, 2014; Pelosi et al., 2007; Schiaffino and Mammucari, 2011; Matheny and Adamo, 2010). This switch is consistent with the view that miRs 206, 29, and 1a bind to Hdacs that reduce the binding affinity of MyoD and Mef2, permitting these TFs to initiate myogenic regeneration. Given that IGF-activated Akt1 modulates p300 to associate with MyoD (Serra et al., 2007), it is intriguing to hypothesize how other parallel regulatory schemas of this signaling pathway alter fundamental changes in TF binding and chromatin states that drive muscle regeneration after severe trauma. Collectively, these experiments imply that muscle repair and regeneration uses different sets of transcriptional programs, ncRNAs, and combinations of TFs as well as chromatin remodeling factors to precisely execute stage-specific gene expression programs. We envision that a wider combinatorial interrogation of such a dataset can represent a valuable resource to extend the networks acting in such a complex microenvironment like the cis-regulatory modules engaged by TFs, miRNAs, and lncRNAs.
    Experimental Procedures
    Introduction The dual ability of human pluripotent stem topotecan (hPSCs) to self-renew and differentiate into any cell type in the body makes them a promising cell source for regenerative medicine, disease modeling, and drug discovery (Takahashi et al., 2007; Thomson et al., 1998). Such applications necessitate the maintenance of large numbers of undifferentiated, genetically stable cells. However, hPSCs are subject to genetic changes in vitro and in the presence of selection pressures, the variants with changes that allow for improved growth outcompete their neighbors and overtake the culture (Draper et al., 2004; Olariu et al., 2010). The commonly observed genetic changes in hPSCs are non-random and involve gains of either parts or whole chromosomes 1, 12, 17, and 20 (Amps et al., 2011; Taapken et al., 2011), indicating that genes within these regions confer selective advantage to variant cells (Avery et al., 2013; Blum et al., 2009). Genetic aberrations that arise in hPSCs during culture can affect their behavior and confound experimental results. Some of the variant cells with common genetic changes show signs of neoplastic progression (Werbowetski-Ogilvie et al., 2009), including reduced apoptosis (Avery et al., 2013; Yang et al., 2008), growth-factor independence (Werbowetski-Ogilvie et al., 2009) and higher cloning efficiency (Barbaric et al., 2014; Enver et al., 2005). Genetic changes can also affect the differentiation propensity of hPSCs. For example, a culture-adapted H7 line displayed a reduced tendency for differentiation to endoderm (Fazeli et al., 2011). Similarly, variant cells with a gain of chromosome 20q11.1-11.2 showed differences in the hematopoietic and neural differentiation protocols compared with their wild-type controls (Werbowetski-Ogilvie et al., 2009). Altered patterns of differentiation caused by accrued genetic changes may significantly affect the use of such cell lines in applications that require the production of differentiated derivatives. Furthermore, the commonly observed genetic changes in hPSCs are also frequently observed in embryonal carcinoma cells, the stem cells of malignant germ cell tumors termed teratocarcinomas (Harrison et al., 2007). Indeed, gain of chromosome 12p is used as a diagnostic marker for testicular germ cell tumors (Sandberg et al., 1996). With hPSCs derivatives entering clinical trials, a possibility that genetic changes may confer malignant properties to hPSCs or their differentiated progeny is a cause of regulatory concern (Goldring et al., 2011). Consequently, scientists using hPSCs need to be vigilant to monitor the cultures for the presence of genetic changes. This necessitates a good understanding of the sensitivities of different methods used for screening hPSC cultures, as preparations of cells declared “normal” and “free of genetic variants” according to a particular methodology may nevertheless harbor variant cells below the level of sensitivity.