MYB bi-allelic targeting abrogates primitive clonogenic progenitors while the emergence of primitive blood cells is not affected.

MYB is a key regulator of definitive hematopoiesis and it is dispensable for the development of primitive hematopoietic cells in vertebrates. To delineate definitive versus primitive hematopoiesis during differentiation of human embryonic stem cells, we have introduced reporters into the MYB locus and inactivated the gene by bi-allelic targeting. To recapitulate the early developmental events more adequately, the mutant and wild type human embryonic stem cell lines were differentiated in defined culture conditions without the addition of hematopoietic cytokines. The differentiation of the reporter cell lines demonstrated that MYB is specifically expressed throughout emerging hematopoietic cell populations. Here we show that the disruption of the MYB gene leads to severe defects in the development and proliferation of primitive hematopoietic progenitors while the emergence of primitive blood cells is not affected. We also provide evidence that MYB is essential for neutrophil and T cell development and the upregulation of innate immunity genes during hematopoietic differentiation. Our results suggest that the endothelial origin of primitive blood cells is direct and does not include the intermediate step of primitive hematopoietic progenitors.

Generation of RUNX1-null reporter human embryonic stem cell line GIBHe008-A.

RUNX1/AML1/CBFA2 (runt-related transcription factor 1/acute myeloid leukemia 1 protein/core-binding factor subunit alpha-2), is a transcription factor that plays a critical role in the development of normal hematopoiesis. RUNX1 is also essential for the development of immune cells and sensory neurons. Chromosomal translocations involving the gene have been associated with several types of leukemia. To investigate the role of RUNX1 in human hematopoietic development we generated RUNX1-null human embryonic stem cell reporter line GIBHe008-A by TALEN mediated homologous recombination. This cell line GIBHe008-A was subjected to detailed characterization by standard assays for human pluripotent stem cells. It provides an ideal model to study the role of RUNX1 in the hESC-derived developmental models.