Abstract

Hox genes are important regulators of normal hematopoiesis, where each step in differentiation from stem cell to differentiated progeny is associated with a particular Hox expression pattern. In addition, abnormal or disregulated Hox gene expression is often associated with leukaemia. In order to understand how Hox gene transcriptional networks govern normal and leukemic hematopoiesis, we have generated ES cell lines in which expression of representative Hox genes can be induced with doxycycline during in vitro differentiation. Previous work showed that the relatively 3′ Hox gene, HoxB4, promotes self-renewal of hematopoietic stem cells, as well as enabling a primitive to definitive hematopoietic transition. We report here the generation of an ES cell line with inducible expression of a relatively 5′ Hox gene, HoxA10. When expressed during the time at which the hematopoietic lineage first segregates from the endothelial lineage (day 4 of embryoid body (EB) differentiation), HoxA10 dramatically alters the immunophenotype of the EB hematopoietic compartment. Whereas 100% of hematopoietic colony forming cells (CFC) from a wild-type day 6 EB are double positive for c-kit and CD41, HoxA10 expression from day 4 to day 6 eliminates the CD41-positive population, while increasing the c-kit-positive population. CFCs were present at relatively normal numbers. In contrast, HoxB4 did not dramatically alter either the frequency or the immunophenotype of this early hematopoietic population, but significantly enhanced the frequency of mixed-lineage CFCs. We further compared the ability of these two Hox genes to promote the outgrowth of an undifferentiated hematopoietic population on OP9 from day 6 EBs. Whereas HoxB4 induced a rapid expansion of this population, concomitantly with ongoing myeloid commitment, HoxA10 induced a much slower outgrowth characterized by a prominent B-lymphoid component as well as myeloid and erythroid elements. We are currently assaying this HoxA10-induced hematopoietic population for adult repopulating activity.

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