C/EBPα bypasses cell cycle-dependency during immune cell transdifferentiation

Our earlier work has shown that pre-B cells can be converted into macrophage-like cells by overexpression of the transcription factor C/EBP alpha or C/EBP beta with high efficiency. Using inducible pre-B cell lines, we have now investigated the role of cell division during C/EBP-induced reprogramming. The majority of cells reprogrammed by C/EBP alpha incorporated BrdU before arresting at G(0), and all C/EBP beta-induced cells incorporated the compound. This contrasts with reports from other systems where transdifferentiating cells essentially do not divide. Although inhibition of DNA synthesis led to an impairment of C/EBP alpha-induced transdifferentiation, sorted G(0)/G(1) and G(2)/M fractions showed no significant differences in their reprogramming kinetics. In addition, knocking-down p53 did not accelerate the transdifferentiation frequency, as it has been described for reprogramming of induced pluripotent (iPS) cells. Time-lapse experiments showed that, after C/EBP alpha induction, approximately 90% of cells divide once or twice, while 8% do not divide at all before acquiring a macrophage phenotype, supporting our BrdU incorporation results. Importantly, the non-dividing cell subset expressed the highest levels of C/EBP alpha and was the fastest in differentiating, suggesting that high levels of C/EBP alpha accelerate both the switching process and the cells' growth arrest. Our data show that traversing the cell cycle is not strictly required for pre-B cell to macrophage conversion and provides new evidence for the notion that the mechanisms of transcription factor induced transdifferentiation and iPS cell reprogramming differ.