Figure 4.

Metabolic shift and the expression of genes regulating reprogramming in fibroblasts grown in N or Ax medium. (A): OCR during basal respiration, displayed as AUC, of human hFs 3 and hFs 4 grown in N or Ax medium (B): OCR in the presence of glucose as sole energy substrate, displayed as AUC, of human somatic fibroblasts hFs 3 and hFs 4 grown in N or Ax medium. (C): Western blot analysis of HIF1α, PDK1, PKM2, and GLUT1 in hFs 3, hFs 4, and hFs 5 cultured in N or Ax medium. β-Actin was used as an internal control. (D): A proposed model for the effects of high cell proliferation and insulin signaling on reprogramming of human fibroblasts. In fibroblasts with normal proliferation, cells maintain higher glycolysis and low OXPHOS. Cells cultured in Ax medium, with higher cell proliferation, exhibit increased growth factor (insulin/IGF-1) signaling and a higher OXPHOS by downregulating the expression of HIF1a, PDK1, PKM2, and GLUT1 proteins, leading to a significant decrease in the efficiency of induction of pluripotency (hiPSCs). All experiments were performed three times, represented as mean ± SD. Statistical significance was determined by Student’s t test. ∗, p < .05; ∗∗, p < .01 for Ax versus N. Abbreviations: AUC, area under the curve; GLUT1, glucose transporter-1; Ax, AminoMAX medium; HIF1α, hypoxia inducible factor-1 α; hiPSC, human induced pluripotent stem cell; N, Dulbecco’s modified Eagle’s medium; OCR, oxygen consumption rate; OSKM, OCT4, SOX2, KLF4, and cMYC; OXPHOS, oxidative phosphorylation; PDK1, phosphoinositide dependent kinase-1; PKM2, pyruvate kinase M2 isoform.