Preparation of Induced Pluripotent Stem Cells from Fibroblasts under Circadian Rhythm

Abstract

iPSCs are conventionally generated under constitutive promoters for a constant expression of pluripotency factors, yet the yield of reprogrammed cells is low. Since somatic cells are entrained in a circadian rhythm that regulates their metabolic and physiological functions, we hypothesize that the expression of the reprogramming factors should be done under circadian rhythm instead of a continuous expression. Doing this might hasten the process and improve the yield of iPSCs. To test this, I prepared plasmid constructs with the OSKM reprogramming cassette attached downstream of the circadian promoters Per2 or Bmal1. However, I could not obtain successful midipreps of these plasmids due to time constraints. Nevertheless, iPSCs were generated from fibroblasts with plasmids containing the OSKM genes under the constitutive LTR promoter. This independent experiment helped me become familiar with the process of virus generation and transduction and to determine the optimum seeding density to achieve iPSC formation from fibroblasts.