Reprogramming of Mouse Fibroblasts to an Intermediate State of Differentiation by Chemical Induction

Abstract

Induced pluripotent stem cells (iPSCs) generated by forced expression of four transcription factors offer promises for regenerative and therapeutic uses in human diseases. However, it is necessary to overcome the risk of tumorigenicity caused by the use of potent oncogenes and the use of randomly integrating vectors before the iPSC technology can be applied to human medicine. Stem cells and cancer cells share many features in common, implying that there are similar underlying mechanisms in their development. Small molecules have been used to induce cell reprogramming for lineage trans-differentiation and for maintaining pluripotency of stem cells. In this study, we investigated the possibility of replacing all reprogramming viral factors with small molecules. To this end, we evaluated the effects of carcinogens at nongenotoxic levels on somatic cells. We identified 16 candidate chemicals through biology-oriented in silico high-throughput screening with commercially available inventories from Sigma-Aldrich for cancer research, and established a reprogramming protocol of 16-day treatment followed by 5 days of recovery. This protocol was applied to B6/129 mouse embryonic fibroblasts (MEFs) at passage 3. From recovery day 2, colonies appeared at an efficiency of 0.02%. These colonies were positive for both alkaline phosphatase and surface specific embryonic antigen-1 (SSEA-1) at a comparable level to those of mouse embryonic stem cells (ESCs). Global gene expression analysis with a 38K gene MEEBO microarray revealed that the colonies had 564 (1.5%) differentially expressed genes compared to MEFs at day 0 of treatment, and these genes were enriched in "neuromuscular differentiation.'' Moreover, 122 differentially expressed genes in the colonies were ESC-enriched, including downregulated somatic markers and upregulated stem cell markers. In conclusion, combined chemical treatment of MEFs herein might have caused these cells to transverse to an intermediate state within the mesodermal lineages.