Stem Cell : Programming and Reprogramming

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Thomas Graf

Thomas GrafOur laboratory focuses on understanding the mechanism by which C/EBPa induces the transdifferentiation of B cells into macrophages. Using an inducible form of C/EBPa this system permits to convert 100% of pre-B cells into macrophages in a matter of days. 

We have recently asked whether primary pre-B cells on their way to become macrophages go through intermediate stages that correspond to early progenitors, or short of that, whether gene expression programs characteristic of progenitors are transiently activated. Using a variety of approaches we found that there is no overt retrodifferentiation and that the process, with small exceptions, is essentially direct. Our gene expression data also show that the resulting macrophages closely resemble normal macrophages, but in addition downregulate a large number of cell cycle genes.

We have also studied genome-wide changes in DNA methylation and found, in collaboration with the laboratory of Esteban Ballestar, that there are surprisingly few changes occurrying during the switch from B cells to macrophages. However, knockdown experiments with Tet2 showed that about 6% of the genes that become upregulated after C/EBPa induction depend on Tet2 for full activation. Tet2 binds to the promoters of these target genes and catalyzes the conversion of methylated cytosine residues into hydroxylated cytosines, correlating with the derepression of the corresponding target gene. In addition, these modified residues become subsequently demethylated. The observed defective derepression of macrophage restricted genes in Tet2 knockdowns may reflect the block of differentiation in acute myeloid leukemia and other hematopoietic neoplasms in which Tet2 is inactivated by deletions or mutations. 

Finally, we have asked whether it is possible to reprogram human B cells into macrophages, and have tested EBV-Myc immortalized Burkitt's lymphoma cells, which correspond to mature B cells. Using an inducible form of C/EBPa we found one line (Seraphina) that can be switched with either estrogen or tamoxifen to macrophages at nearly 100% efficiency. Over the course of 8 days these cells exhibit >2fold changes in about 11,000 genes, including many well known markers and transcription factors characteristic for B cell and macrophages. The resulting cells became adherent, large and acquired phagocytic capacity, and also stopped dividing after one population doubling. These results show that it is, in principle, possible to transdifferentiate human cells at high efficiencies. Interestingly, Seraphina cells induced to transdifferentiate lose their tumorigenic potential in immunodeficient mice, raising the possibility that small molecules might be found that mimic this effect and that could be of therapeutic use for the treatment of patients with lymphomas. 

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Supporting publications

Cell
Cell : Stem Cell
Trends in Pharmacological Sciences
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