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  • In conclusion we expect that this VP


    In conclusion, we expect that this VP64dCas9-BFPVP64 platform, which is significantly more potent than previous versions, will encourage the adoption of CRISRP/Cas9-based TF technology to achieve multiplexed gene activation for reprogramming as well as nonreprogramming applications in basic science, biotechnology, and medicine.
    Experimental Procedures
    Introduction Factor-mediated reprogramming, the process by which overexpression of a defined set of transcription factors converts one cell type into another, has important implications for regenerative medicine and demonstrates the power that transcription factors have as cell fate determinants (Jaenisch and Young, 2008). This has been shown for pluripotent stem cells, where three transcription factors (Oct4, Sox2, and Klf4) are sufficient to induce any cell type to become induced pluripotent stem cannabinoid receptors (iPSCs) that are transcriptionally, epigenetically, and functionally indistinguishable from embryonic stem cells (ESCs) (Takahashi and Yamanaka, 2006; Wernig et al., 2008a). Ectopic expression of key transcription factors in somatic donor cells has been used to generate many different cell types, including cells resembling blood cells (Heyworth et al., 2002; Xie et al., 2004), brown fat cells (Kajimura et al., 2009), hepatocytes (Huang et al., 2011), Sertoli cells (Buganim et al., 2012), and various cell types of the neural lineage (Vierbuchen et al., 2010; Kim et al., 2011; Son et al., 2011; Yang et al., 2013). An important advance has been the generation of neural stem cells (NSCs) from embryonic fibroblasts, as these self-renewing somatic stem cells can be expanded for use in clinical application (Lujan et al., 2012; Han et al., 2012; Ring et al., 2012). However, a number of issues remained unresolved and are the focus of this paper. In these studies, the induced NSCs (iNSCs) were still dependent on the constitutive factor expression. Importantly, because the published studies used a heterogeneous population of mouse embryonic fibroblasts (MEFs) as starting cells, it has been difficult to ascertain that indeed nonneural somatic cells gave rise to the iNSCs rather than preexisting neural cells (such as neural crest cells) present in the donor population. Finally, an extensive epigenetic analysis has not been performed on iNSCs, or any other directly converted cell type, to determine whether the chromatin has been reset to allow for cell type-specific gene expression to persist in the absence of the exogenous factors. Here we show that transient overexpression of 8 transcription factors—Brn2, Hes1, Hes3, Klf4, Myc, Notch1, (NICD), PLAGL1, and Rfx4—in fibroblasts generates iNSCs that can differentiate into neurons, astrocytes, and oligodendrocytes and have similar genome-wide gene expression patterns and enhancer usage as primary-derived control NSC lines. We then used iPSC technology to generate a genetic system that converts multiple adult cell types to tripotent iNSCs, including liver cells and B cells that carry rearrangements in the immunoglobulin loci. Our results indicate that specific combinations of transcription factors can reset the genome of diverse cell types to that of NSCs.
    Discussion Adult somatic cells have been directly reprogrammed to mature neurons (Vierbuchen et al., 2010; Marro et al., 2011; Son et al., 2011). However, since these cells are postmitotic, it is difficult to obtain large numbers of cells required for transplantation therapy. The direct conversion of readily accessible adult somatic cells to NSCs is of clinical significance since NSCs can proliferate and thus allow the generation of large quantities of neurons and glia that could be used for eventual transplantation therapy.
    Experimental Procedures
    Author Contributions
    Introduction Saliva, the secretion of the salivary gland (SG), crucially maintains the physiological balance in the oral cavity and initiates food digestion. Like many other cannabinoid receptors organs, SGs undergo cell renewal, presumably enforced by a small pool of stem cells. Dysfunctional SG homeostasis may be caused by improper SG stem cell functioning, leading to disease. Disease-induced hyposalivation often leads to xerostomia, with symptoms including dry mouth/nasal passages, sore throat, loss of oral hygiene, dental caries, oral candidiasis, loss of taste, and difficulties with swallowing and speaking, which collectively reduce the patient’s quality of life (Vissink et al., 2010). Hyposalivation can be a consequence of autoimmune disorders (Sjögren syndrome), endocrine disorders (diabetes mellitus and hyper-/hypothyroidism), neurologic disorders, or radiation damage in head and neck cancer patients after radiotherapy.