## 3 Results and Discussion  
It is already documented that shear stress conditions and vitamin C aid in transgene-integrated generation of iPSCs. However, we alternatively induced stem cell-like cells using external stimuli without integrating OSKM genes required for iPSC induction. Figure 1 provides the schematic illustration of the induction methods used in this study for generating stem cell-like cells in suspension culture. We used an electronic pipette to produce short-term shear stress (STSS) and compared its results to induce spheroid formation from MEF cells with the group that was subjected to shear stress together with vitamin C (SSVC) treatment. Subsequently, the spheroids were characterized for stemness and potential to differentiate into different cell types. Figure 2a and Figure S1 show the morphology of spheroids induced by STSS and SSVC from MEF. The area of spheroids was increased over time in both of the treatments by STSS and SSVC. After picking each spheroid, they were measured for length and area. The spheroids of the SSVC group tended to be longer and larger than that of the STSS group (Figure 2b,c), suggesting that SSVC has high efficiency for generating cells per spheroid. Alkaline phosphatase (ALP) staining was performed to identify the early marker of iPSC (Figure 2d). The MEF spheroids cultured under SSVC responded stronger with ALP solution than that of the STSS group. These data showed that the SSVC technique was more likely to form spheroids than STSS technique, as well as increase the possibility of reprogramming. Thus, we draw a hypothesis that MEF spheroids, which have been subjected to shear stress, gain potential for cellular reprogramming and that it is further enhanced with the addition of vitamin C.  
To identify pluripotent characteristics of MEF spheroids, we analyzed the expression of Oct4 and SSEA-1 by immunocytochemistry (ICC) (Figure 3a,b). Both the SSTS- and SSVC-treated groups showed the presence of pluripotency markers in comparison to negligible expression in the MEF control group. These results suggest that both groups have properties of stem cells as indicated by the ALP analysis results. However, no significant difference was observed between the two groups in ICC. For quantification of expressed pluripotent markers, Western blot assay was performed (Figure 3c). The Western blot results clearly determine the increased expression level of Oct4 and SSEA-1 in the SSVC experimental group compared to the STSS group, suggesting that addition of vitamin C to shear-stress-induced cells, as in the SSVC method, enhances the expression of pluripotent stem cell markers more than the SSTC method. Finally, we determined the differentiation potential of MEF spheroids induced by SSVC method (Figure 4). To validate the differentiation capability, Neuronal Class III b-Tubulin (Tuj1), which is a neuronal marker of the ectoderm, sarcomeric alpha-actinin, which is a cardiomyocyte marker of the mesoderm, and alpha 1 feptoprotein, which is one of the endoderm markers, were used. The SSVC-induced spheroids were observed to be partially differentiated into neuron, mesoderm, and endoderm. These results showed that shear stress and vitamin C together induced partial reprogramming. In summary, we showed that the SSVC method enhances spheroid formation and expression of pluripotent stem cell markers more than the STSS method. In other words, shear stress and vitamin C caused a synergistic effect in inducing stem cell-like cells. However, we observed that only a small fraction of the spheroids induced by SSVC differentiated into specific cells. Therefore, we conclude that spheroids formed by various external stimuli were stem cell-like cells, not induced pluripotent stem cells, and could suggest the possibility of direct conversion from the fibroblast. Figure 2: Comparative induction of MEF (mouse embryonic fibroblasts) into stem cell-like cells by STSS and SSVC methods in comparison to the control MEF. (**a**) Morphology of induced spheroids (scale bars = 200 \(\upmu\)m).