Stepwise reprogramming of B cells into macrophages
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Current schemes of adult hematopoietic differentiation show an early branching of multipotent progenitors into myeloid and lymphoid precursors. However, several lines of evidence indicate a developmental connection between B lineage cells and macrophages. In an attempt to learn the rules that govern the myeloid/lymphoid cell dichotomy we have asked whether it is possible to reprogram B lineage cells into macrophages by enforced transcription factor expression.;For this purpose we expressed C/EBPalpha by retrovirus infection in purified CD19+ bone marrow B lineage cells. The infected cells were cultured in vitro and their fates were examined over time by FACS. We observed a down-regulation of CD19, a B cell marker, and a concomitant up-regulation of Mac-1, a myeloid marker, within 2 days after infection and 60-70% of the cells became CD19-Mac-1+ within 4-5 days. The reprogrammed cells resembled macrophages by the expression of macrophage (but not neutrophil) genes and the down-regulation of all B cell genes tested, including the three key B cell transcription factors E2A, EBF and Pax5. The reprogrammed cells morphologically resembled macrophages and more importantly they were phagocitic. The B cell origin of the reprogrammed cells was confirmed by their harboring of immunoglobulin gene rearrangements.;Experiments indicate that the reprogramming requires PU.1. In double infection experiments, PU.1 synergized with C/EBPalpha in reprogramming B cells into macrophages. In addition PU.1 reprogrammed all "C/EBP-non-responders" into macrophages. The latter observation indicates a threshold level of endogenous PU.1 expression required for C/EBPalpha induced B cell to macrophage reprogramming. In addition our experiments suggest that Pax5, the so called B cell commitment factor, is a direct target of C/EBPalpha by reporter assays using the Pax5 responsive CD19 promoter driving the expression of the luciferase gene. Recent evidences suggest that the reprogramming complex is more complex, showing that elevated levels of PU.1 suppress the expression of EBF and thereby other early B cell genes.;In summary, we have shown that C/EBPalpha is able to reprogram B cells to become macrophages by synergizing with endogenous PU.1 to activate the myeloid genes on the one hand and by inhibiting Pax5 on the other hand, leading to the down-regulation of B cell genes. This principle may also apply to normal hematopoietic development, such as in the fetal liver where a bipotent B cell macrophage progenitor is known to exist.