Fig 2: Plasmid construction and mRNA synthesis. (a) Successful amplification of the four factors from selected organs using specific primers for each. Oct4 (O) and Sox2 (S) were amplified from testes while c-Myc (C) was amplified from small intestine. Klf4 (K) was correctly amplified from colon. The DNA marker (M) used was a 2000 bp marker; (b) The eukaryotic expression vector (pCDNA 3) was digested using the same enzymes used for cutting the genes. Lane (1) shows the result of double restriction of the plasmid using EcoRI and XhoI, while Lane (2) revealed the single cut using XhoI. Lane (3), shows the uncut circular plasmid. (M): DNA marker (5000 bp); (c) Confirmation of the successful cloning through digestion of the newly formed recombinant plasmids (pCDNA–Oct4, pCDNA–Sox2, pCDNA–cMyc and pCDNA–Klf4) using EcoRI and XhoI enzymes resulted in two bands that were similar to the expected size of each gene as showed in Lane (2), while Lane (1) shows the single cut of each recombinant plasmid (M): DNA marker (5000 bp).

Fig 3: Ectopic Schwann Cell Migration in the Nerve Bridge of Sox2 KO Mice and Sox2 Regulating Robo1 Expression in SCs(A) Schwann cell (GFP+) migration from both proximal and distal nerve stumps in control mice 6 days after sciatic nerve transection injury.(B) Ectopic Schwann cell migration (white arrows) in the nerve bridge of Sox2 KO mice 6 days after transection injury.(C) Higher magnification image from (B, dotted-line square) showing regenerating axons (labeled with neurofilament, red, indicated by arrowheads) following the ectopic migrating Schwann cells (white arrows) and leaving the nerve bridge.(D) Schwann cells stayed in the nerve bridge in control mice at 14 days following sciatic nerve transection injury.(E) Ectopic migrating Schwann cells (white arrows) leaving the nerve bridge in Sox2 KO mice at 14 days after injury.(F) Ectopic migrating Schwann cells (white arrows) localizing in front of regenerating axons (indicated by arrowheads) of Sox2 KO mice.Scale bar in (A, B, D and E) represents 200 µm, in (C) represents 60 µm, and in (F) represents 30 µm.(G and H) Microarray data (G) and RT-PCR (H) from control and Sox2-overexpressing Schwann cells. Values in (G) represent the fluorescence intensity of dye-labeled cDNA fragments that have hybridized to the probes on the microarray chip.(I) qRT-PCR validation of Robo1 mRNA upregulation in Sox2-overexpressing Schwann cells, n = 3.(J) Quantification of Robo1 protein levels in the distal nerve stump of control (Con) and Sox2 KO mice at 4 and 7 days after injury, n = 3.(K) Western blots showing Robo1 and Robo2 protein upregulation in Sox2-overexpressing Schwann cells.(L) Western blots comparing Robo1 and Robo2 protein levels in the distal nerve stump of control and Sox2 KO mice at 4 and 7 days after injury.(M) Western blot comparing Robo1 protein levels in the distal nerve stump of control (Con) and Sox2-overexpressing (OE) mice at 7 days following injury.(N) Quantification of Robo1 protein levels from (M), n = 3.(O) RT-PCR showing that Slit3 is highly expressed in the nerve bridge at 7 days after injury; dorsal root ganglion (DRG) samples have been used as positive controls.** in (I), (J), and (N) indicate p < 0.01 compared with controls.Several z series were captured on a Zeiss LSM510 confocal microscope in (A), (B), (D), and (E), covering the entire field of interest. The individual series were then flattened into a single image for each location and combined into one image using Adobe Photoshop software (Adobe Systems).

Fig 4: Characterization of the mRNA induced pluripotent stem cells (iPSCs). (a) The morphological characteristics of iPSCs with round ES-like colonies were distinguished by tightly defined borders and a high nuclear/cytoplasm ratio (magnification 40×); (b–d) showed positive alkaline phosphatase activity of the mRNA iPS either grossly (b, magnification 10×) or through microscopic observation (c, magnification 100×) and (d, magnification 200×); (e) Total RNA was isolated from three mRNA iPS clones and non-transfected MEF cells were used for RT-PCR to detect expression of pluripotency markers: Oct4, Sox2, Nanog, Rex1, Klf4 and cMyc. Results showed that iPS colonies expressed all markers in contrast to MEF cells; (f) Detection of the methylation status of Nanog and Oct4 promoters in both MEF cells, iPS and ESCs by bisulfite sequencing revealed a high percentage of de-methylation in both promoters compared to the parent MEF cells. Open circles indicate the un-methylated state and dark, filled circles indicate the methylated state.

Fig 5: Changes in introduced factors and pluripotency markers during reprogramming. Quantification of pluripotency factors expression by qRT-PCR at different times (D1, D6, D9, D12 and D15) throughout the reprogramming timeline. (a) Total and endogenous expression levels of the transfected factors; Oct4, Sox2, c-Myc and Klf4; (b) the expression level of non-transfected factors (Nanog and Rex-1); (c) Immunostaining of non-transfected factors at day 6 confirmed the results of the qRT-PCR concerning Nanog and SSEA-1, as they showed nuclear localization of Nanog and surface expression of SSEA-1. Nuclei were counterstained with DAPI; (magnification 100×).