Figure 5.

The differential effects of NTF1 and PD0325901 on the choice of programming process at the onset of iPSC induction (A) The quantitative analysis of iPSC colony formation under various treatments. The number of the OCT4-GFP-positive clones is indistinguishable between MEF cells treated with NTF1+PD0325901 and those treated with NTF1 alone on the 7th day postdoxycycline induction. However, the NTF1 alone condition maintained the OCT4-GFP-positive clone number observed on the 7th day throughout the entire course of induction (green bar). In contrast, the NTF1+PD0325901-treated group showed an increase the OCT4-GFP-positive clone number until the end of induction (orange bar). (B) The curve-fitting data of the NTF1 (green) and NTF1+PD (orange) groups extracted from panel A. The NTF1-treated group (green) represents the deterministic process, whereas the NTF1+PD group (orange) shows the stochastic model. (C) Representative time-lapse photographs of iPSC^{Col1a1 4F2A Oct4-GFP} clone formation induced by NTF1+PD0325901. The series of photographs was obtained from the same clone from the 9th to 15th day postdoxycycline induction. The scale bar denotes 50 µm. (D) The selection of a reprogramming process depends on the pSTAT3 vs. pErk1/2 ratio. In the presence of NTF1, the pSTAT3/pErk1/2 ratio creates a unique cell state and a threshold where it engages the deterministic process during reprogramming. If the ratio is higher than the NTF1-established ratio, reprogramming will occur through the stochastic route instead. (E) A proposed mechanism addresses the selection of the somatic reprogramming mode. The current model represents an example in which the surrounding microenvironment imposed upon MEF^{Col1a1 4F2A Oct4-GFP} cells changes the cell fate at the onset of iPSC formation. Extraneous E-cadherin transduces the signal through EGFR/ErbB2 and LIFR/gp130 resulting in activation of the downstream effectors STAT3 and Erk1/2. As the activities of pSTAT3 and pErk1/2 counteract each other, the net output through the respective down- and upregulation of Snail2 and E-cadherin overcomes the MET barrier. In this scenario, pErk acts as a negative regulator on the entry of reprogramming. Accordingly, the balance between STAT3 and Erk1/2 activity is thus essential in setting the stage for cell fate at the entry point of somatic reprogramming. (F) In addition to controlling the entry, the balance between STAT3 and Erk1/2 activity also regulates the choice of entry model, where either the deterministic or stochastic process could be chosen at the onset of reprogramming. The presence of NTF1 in the surrounding environment alters the ratio of pSTAT3 and pErk activity, resulting in adoption of the deterministic model. In contrast, the increment of STAT3 activity, along with the deactivation of Erk, engages the stochastic model in the presence of NTF1+PD0325901 for the first three days of induction. Previous reports have supported that either deterministic or stochastic processes could be adopted during reprogramming depending on the source of the cell type. In the present study, our evidence supports that different modes of operation can act on the same MEF cells and are interchangeable via modulation of the STAT3 and Erk activity. n = 3. PD: PD0325901; WP: WP1066. *, p < 0.05; **, p < 0.005; ns, not significant.