Many SMs have been reported to facilitate reprogramming

Many SMs have been reported to facilitate reprogramming. For example, sodium butyrate (N, NaB), a histone deacetylase (HDAC) inhibitor, greatly improved reprogramming efficiency by upregulating epigenetic remodeling and pluripotency-associated fasudil (Mali et al., 2010; Zhang and Wu, 2013). In another study, Yu et al. (2011) reported that an SM cocktail containing MEK inhibitor PD0325901 (PD), GSK3β inhibitor CHIR99021 (C, Chir), transforming growth factor β (TGF-β)/Activin/Nodal receptor inhibitor A-83-01 (A), ROCK inhibitor HA-100, and human leukemia inhibitory factor (hLIF), facilitates iPSC generation from human neonatal foreskin fibroblasts. Also, Chir was used to promote iPSC generation from human primary keratinocytes (Li et al., 2009). A summary of these SMs is presented in Table 3. In addition, reprogramming factors OSKM have been shown to trigger cell senescence by upregulating P53, P16, and P21 (Banito et al., 2009). P53-mediated cell senescence has been known as a barrier to reprogramming (Hong et al., 2009; Kawamura et al., 2009) and downregulation of P53 could promote reprogramming and iPSC generation (Okita et al., 2011). Cyclic pifithrin-a (P, CPFT-a) is a transcriptional inhibitor of P53. Compared with P53 short hairpin RNA or dominant-negative P53 protein, inhibition of P53 by CPFT-a is efficient and easy to control. Because P53 plays an important role in safeguarding genome stability, it is ideal to release its inhibition in newly generated iPSCs after reprogramming. Here, to enable efficient iPSC generation from hUCs with various proliferation capabilities, we optimized non-viral approaches for hUC reprogramming. Firstly, we selected CPFT-a, a P53 inhibitor, and other SMs known to promote reprogramming such as A-83-01, Chir, thiazovivin (T, Tzv), and NaB, to form an SM cocktail. Aided by this SM cocktail and another SM, PD0325901 introduced only at later stage of reprogramming, the hUC reprogramming efficiency was significantly enhanced, up to 170-fold. The highly efficient reprogramming could be achieved in 2 weeks with the aid of these SMs. Furthermore, we found that non-transfected autologous hUCs can be used as feeder to overcome the cell death caused by electroporation for the transfection of reprogramming genes. Upon these optimizations, successful rates of generating viral-free iPSCs from hUCs with different states were greatly improved.
Results
Discussion The role of SMs in aiding iPSC generation has been extensively examined in different somatic cells, such as human adult/fetal fibroblasts, mouse embryonic fibroblasts, and human epidermal keratinocytes (Esteban et al., 2010; Hou et al., 2013; Li et al., 2009; Lin et al., 2009; Mali et al., 2010; Shi et al., 2008; Yu et al., 2011; Zhu et al., 2010). Here, we re-examined the role of different SMs in hUC reprogramming. We found that Chir and CPFT-a significantly improved hUC reprogramming (p < 0.001), while NaB showed moderate improvement (p < 0.05). In contrast, A-83-01 and Tzv had a minimal effect when they were used alone (Figure S2). PD0325901 has been shown to suppress the growth of non-reprogrammed cells and promote reprogramming when added at a later stage (Shi et al., 2008). Consistently our results show that during hUC reprogramming, adding PD0325901 at an early stage inhibits reprogramming but at a later stage promotes reprogramming (Figures S3A–S3D). Among these six tested SM, CPFT-a, a P53 inhibitor, has not been previously reported to be used for reprogramming. has been reported to be a critical barrier during somatic cell reprogramming (Marion et al., 2009) and also an important tumor suppressor (Levine and Oren, 2009). For efficient reprogramming, P53 is usually a considered target for suppression during iPSC generation. In fact one of the reprogramming factors, SV40LT, is known as a negative regulator of P53. In addition, suppressing P53 through other biological approaches such as RNAi or even gene knockout promotes reprogramming, but raises safety concerns regarding genome instability due to the low activity of P53. Therefore, considering that SV40LT, another negative factor for P53, was already included for reprogramming, we minimized the application of CPFT-a during hUC reprogramming. Adding a low concentration of CPFT-a indeed suppressed P53 expression and enhanced the final efficiencies of iPSC generation during hUC reprogramming (Figures 1F and 1G), indicating a synergistic suppression of P53 with SV40LT. Another SM reported for enhancing iPSC generation, NaB, did not show a significant effect in promoting hUC reprogramming (Figure 1L). NaB was known to activate the expression of miR302/367, which further promotes reprogramming (Zhang and Wu, 2013). Liang et al. (2010) reported that NaB facilitated reprogramming only in the presence of exogenous c-Myc. For hUC reprogramming, we replaced C-Myc with miR302/367 as reprogramming factor, which might explain the less significant effect of NaB on hUC-derived iPSC generation. Nevertheless, due to the variation in different batches of hUCs (Figure S2), it is still beneficial to include NaB in the SM cocktail when reprogramming hUCs with poor proliferation.