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    • In the work we present here we investigated how miR

    2018-10-26

    In the work we present here, we investigated how miR-29a influences iPSC generation in mouse and we analyzed canonical WNT signaling pathway and the pluripotency-related active DNA demethylation enzyme Tet1 in this context, both of which are known to be modulated by miR-29. Furthermore, we show evidence that miR-29 affects these pathways in human foreskin fibroblasts as well. We found that miR-29 family members are down-regulated in MEF tizanidine hcl and human fibroblasts during the early phase by OKSM-based reprogramming, compared to OKS-based reprogramming. Moreover, we confirm that transient transfection of miR-29 leads to a strong decrease in Oct4-GFP+ colony number, while inhibition of miR-29a by anti-miR transfection increased the number of obtained colonies. Furthermore, we show that siRNA-based downregulation of confirmed miR-29 target Tet1, results in a decrease in reprogramming efficiency. Moreover, we show to our knowledge for the first time that miR-29 targets Gsk3-β in mouse, and that its transfection at the early phase of OKS-induced reprogramming leads to activation of canonical WNT signaling in MEF cells.
    Materials & methods
    Results and discussion
    Funding information São Paulo Research Foundation - FAPESP (including the project fellowships 2010/02616-0 and 2013/19545-7); National Council for Scientific and Technological Development (CNPq); Funding Authority for Studies and Projects (FINEP), Brazil; German Research Foundation (REBIRTH Cluster of Excellence, EXC 62/2; CRC738 \"Optimization of conventional and innovative transplants\").
    Conflict of interest
    Acknowledgments
    Introduction Spinal cord injury (SCI) is one of the most devastating neurological conditions that often causes severe motor and/or sensory deficits in patients. Current managements such as surgeries and physical therapies could only modestly improve patients\' conditions, and leave many patients wheelchair-bound for the rest of their life. Transplantation of neural stem/progenitor cells (NSCs/NPCs) is a novel therapy and has shown promising results in repair and regeneration of lost neural tissues and restoration of neurological deficits (Sahni and Kessler, 2010; Tsuji et al., 2010; Sareen et al., 2014; Salewski et al., 2015). In most reports, human NSCs/NPCs were derived from either fetal brain, spinal cord (Cummings et al., 2005; Salazar et al., 2010; Lu et al., 2012), or human embryonic stem cells (hESCs) (Keirstead et al., 2005; Sharp et al., 2010). These cell sources often have ethical controversies. In addition, they are allogenic, which cause immune rejection and require life-time immunosuppression. Patient specific induced pluripotent stem cells (iPSCs) could overcome these hurdles as a potential source for cell-based therapy. Generally, iPSCs are produced from patients\' somatic cells such as dermal fibroblasts, keratinocytes, and blood cells by transient overexpression of four transcription factors, OCT4, SOX2, KLF4 and C-MYC (OSKM) (Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Yu et al., 2007). iPSCs share almost identical properties with hESCs with additional advantages. iPSCs possess unlimited self-renewal capacity and have the potential to manufacture pure and homogenous neural progeny populations in large quantities. In addition, iPSCs offer genetically matched autologous cell source, which might omit the necessity of using immune suppression drugs. These characteristics set the basis for iPSCs to be a major promising candidate for cell-based replacement therapy. Many reprogramming methods have been rapidly developed to induce a variety of somatic cell types into iPSCs since its invention. The most classical method is infection with retroviruses or lentiviruses. However, both lentivirus and retrovirus integrate into the genome of cells, while effective and sufficient in basic research, neither is suitable for clinical uses due to potential tumorigenicity risks. To avoid the side effects, non-integrating protocols using episomal vectors, Cre-lox system, piggybac vectors, minicircles, recombinant proteins, messenger RNAs, microRNAs, and small molecules, have recently been reported (Chang et al., 2009; Kaji et al., 2009; Kim et al., 2009; Sommer et al., 2009; Woltjen et al., 2009; Yu et al., 2009; Zhou et al., 2009; Jia et al., 2010; Warren et al., 2010; Anokye-Danso et al., 2011; Rao and Malik, 2012; Hou et al., 2013), which have shown variable yields and reproducibility. Recently, Sendai viruses have been established and shown to be able to reprogram dermal fibroblasts, CD34+ hematopoietic cells and urine derived cells (Fusaki et al., 2009; Ye et al., 2013; Afzal and Strande, 2015; Rossbach et al., 2016). As negative sense RNA viruses, Sendai viruses do not integrate into the genome of human cells and are non-pathogenic to humans (Fusaki et al., 2009; Ban et al., 2011; Macarthur et al., 2012a). Most importantly, unlike several other non-integrating reprogramming methods, the reported reprogramming efficiency of Sendai viruses has been high and consistent (Lieu et al., 2013).