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    • In many studies the reprogramming factors were delivered

    2018-11-02

    In many studies, the reprogramming factors were delivered individually using monocistronic vectors. However, due to differences in vector uptake, expression levels of each gene in each cell are highly variable (Lo et al., 2015). Since the ratio between the factors is one of most critical factors for successful reprogramming (Carey et al., 2011; Kim et al., 2015; Papapetrou et al., 2009), the optimal stoichiometry of the reprogramming factors enhances reprogramming efficiency. To achieve equimolar expression of multiple proteins, genes can be linked with self-cleaving 2A-like sequences of CHYSEL polypeptides, which are used by RNA viruses to separate multiple viral genes to be translated (de Felipe et al., 2006). In this system, cleavage occurs through ribosomal skipping during translation, resulting in the release of the upstream protein while translation of the downstream mRNA continues. Commonly used 2A peptides in research are from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and Thosea asigna virus (T2A) (Yang et al., 2008). As our OS vector linked with E2A can efficiently reprogram hematopoietic chemokine receptor antagonist (Meng et al., 2012; Su et al., 2013a, 2016), we use E2A to link two or more genes to ensure the equimolar expression of several genes in this study. In our previous study, using three EV plasmids encoding OCT4-E2A-SOX2 (OS), BCL-XL (B), and MYC-E2A-KLF4 (MK) (OS + B + MK), we generated 20–30 integration-free iPSCs from 1 × 106 cultured MNCs or ∼1 ml of PB (Su et al., 2013a, 2016). In this study, we report that a simple change in vector combination by using two EV plasmids to deliver M and K (M + K) instead of one (MK) leads to some 100-fold improvement in PB reprogramming. We further demonstrate that OCT4 and SOX2 linked by E2A (OS), but not other combinations such as OCT4-E2A-MYC (OM), OCT4-E2A-KLF4 (OK), and OCT4-E2A-SOX2-E2A-KLF4 (OSK), is the best choice for high-efficiency PB reprogramming.
    Results
    Discussion Integration-free iPSCs hold great promise for clinical regenerative medicine. We have reported that an improved EV reprogramming system leads to a 10- to 100-fold increase in PB reprogramming compared with similar methods developed by other laboratories (Su et al., 2013a). However, the EV is still less efficient than another popular integration-free reprogramming vector system, the SeV. After a systemic investigation of vector combinations, we report in this study that a simple change using two individual vectors to express MYC and KLF4, leads to an additional ∼100-fold increase in PB reprogramming than we previously reported (OS + MK + B) (Su et al., 2013a). The marked improvement can be ascribed to a relatively higher M:K ratio and lower KLF4 expression during the course of reprogramming. Another important factor for successful reprogramming is balanced expression of OCT4 and SOX2 mediated by a bicistronic vector. Other combinations such as OM + S + K + B, OK + S + M + B, or OSK + M + B show a significant decrease in reprogramming efficiency compared with OS + M + K + B, highlighting the importance of vector design. All five factors are critical, in particular O, S, M, and K. The iPSCs are indistinguishable from those generated with the previous protocol in expression of pluripotency markers and teratoma-forming ability. In addition, the iPSC lines show no residual plasmids and a normal karyotype after long-term culture. Stoichiometry of reprogramming factors is one of the most critical factors for successful reprogramming. We report that equimolar expression of O and S leads to a 20- to 40-fold increase in reprogramming compared with monocistronic expression of O and S. This is likely due to inappropriate ratios in the vast majority of cells transfected with two vectors encoding O and S. Superficially speaking, balanced expression of mesoendoderm lineage specifier OCT4 and ectoderm lineage specifier SOX2 permits reprogramming to iPSCs, whereas unbalanced levels of O and S attenuate induced reprogramming to pluripotency (Montserrat et al., 2013; Shu et al., 2013). Mechanistically, O and S, along with NANOG, constitute the core transcriptional regulatory circuitry in iPSCs and embryonic stem cells (Boyer et al., 2005; Chen et al., 2008; Kim et al., 2008). After expression of O and S in transfected cells, they form a heterodimer (Remenyi et al., 2003) to bind the canonical motif, in which the SOX2 binding site is followed immediately by an octamer site (Ng et al., 2012), synergistically activating pluripotency factors like NANOG (Kuroda et al., 2005; Rodda et al., 2005). Multiple studies have also demonstrated that O in concert with S increases the transcriptional activity of OCT4 (Chew et al., 2005; Jang et al., 2012). In addition, more than 400 genes expressed in pluripotent stem cells are bound by both O and S to promote pluripotency and self-renewal (Boyer et al., 2005). All these data collectively provide an explanation for the remarkable increase in reprogramming efficiency mediated by equimolar expression of O and S.