Chemical Induction of Alkaline Phosphatase and Surface-Specific Embryonic Antigen-1 Positive Colonies from Mouse Embryonic Fibroblasts.

Abstract

Induced pluripotent stem cells (iPS) generated by transcription factors offers promises for the development of patient-specific pluripotent cells and for research models of human diseases. However, it is necessary to overcome the risk of tumorigenecity caused by the use of potent oncogenes and the use of randomly integrating vectors before the iPS cell technology can be applied to human medicine. Somatic stem cells and cancer cells share many features in common, and both cell types can self-renew and differentiate. These imply that there are mechanical similarities between these cell types, such as the Wnt and MAPK signaling pathways and pluripotency marker gene expression. Small molecules are attributed to be one of the major contributors of cancer initiation and/or cancer proliferation. In this study, we investigated the possibility to replace all reprogramming transcription factors with small molecules. To this end, we evaluated the effects of non-genotoxic carcinogens on somatic cell reprogramming. We identified 18 candidate chemicals through in silico high-throughput screening (HTS) with commercially available Sigma-Aldrich carcinogen inventory. Among them, we used 16 chemicals based on their physicochemical and biological property such as solubility, activation of molecular targets, and cytotoxicity. We first identified the optimal doses for individual and combined treatments of the selected chemicals through a 3-day range finding study. A reprogramming protocol of 16-day treatment followed by a 5-day recovery period was established. We applied this protocol on B6/129 mouse embryonic fibroblasts (MEF) at passage 3. From recovery day 2, cell colonies appeared at 0.01 to 0.03% efficiencies from 1.5×105 cells plated on the first day of treatment. These colonies were positive for both alkaline phosphatase (AP) and surface specific embryonic antigen 1 (SSEA-1) at a comparable level to those of mouse embryonic stem cells (mES). Semi-quantitative RT-PCR analyses confirmed that these colonies lost the expression of Fibrillin 2, a fibroblast marker, as in the case for mES. Global gene expression analysis with a 38K gene MEEBO microarray revealed that the colonies had 564 (1.5%) differentially expressed genes compared to Day 0 MEF, and these genes were enriched in "apoptosis," "blood vessel development," and "neuromuscular differentiation." In addition, 109 differentially expressed genes in the colonies had mES-like expression patterns, those included down-regulated Col6a3 and Thy1 (somatic markers), and up-regulated Ccnd1 and Gpc3 (progenitor markers). However, clustering analysis of "Stem cell maintenance" related genes showed distinct correlation between the colonies and iPS/mES. Furthermore, metalloproteinase-resistance and active xenobiotic metabolism were chemically induced and recognized as adverse effects. We also failed to generate transferable embryos by 8-cell mouse embryo aggregation with these colonies. In conclusion, combined chemical treatment of MEF herein might transverse these cells into intermediate cells within mesodermal lineages. It cannot be excluded that the expression of the marker genes of mesodermal lineages could be caused by cellular stress rather than cellular transversion. (poster)