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MYB bi-allelic targeting
20, suggesting that the granulocyte differentiation and maturation were inhibited by MYB overexpression that resulted in the accumulation of immature CD11c+CD66blow myeloid cells. The negative effect of Myb overexpression on granulocyte maturation has been observed in the mouse progenitor differentiation model.27 Taken together with the limited recovery of CFU-mix pro- genitors, these data suggest that an excess of MYB dysreg- ulates the hESC-derived hematopoietic development.
Discussion
In this study, we created a model in which the emergence of the early primitive blood cells was effectively uncoupled from the development of the primitive hematopoietic pro- genitors. The ability of differentiated hESC to generate large numbers of blood cells in the absence of cytokines provided an initial idea that early blood cells do not require the proliferation of clonogenic hematopoietic progenitors for their emergence. We have been unable to detect mRNA of EPO, TPO, G-CSF, GM-CSF, and IL3 in differentiating cultures of hESC up to day 12; thereby the hESC-derived erythro-myeloid progenitors are unlikely to proliferate and produce day 6 primitive blood cells.
The conclusive evidence of progenitor independence of early human blood cells was obtained from our gene ablation studies of MYB. Primitive erythroid and CFU- MixP progenitors failed to develop in the absence of MYB and thus could not be a source of the MYB-independent cells of the erythro-megakaryocyte lineage. These data suggest that the primitive clonogenic progenitors and the first wave of primitive blood cells originate from HE inde- pendently (Figure 8). Our findings contradict the conven- tion that all primitive blood cells are derived from primi- tive progenitors.1,3,4 Despite their MYB-independence the early human blood cells specifically upregulated transcrip- tion from MYB promoters. One possible explanation of
this observation is that the absence of the functional MYB protein is compensated by other transcription factors. Alternatively, the MYB expression is an insufficient pre- requisite for turning HE and early blood cells into hematopoietic progenitors. In this view, additional devel- opmental cues select cells for specification into functional progenitors.
We obtained ample evidence that MYB-dependent hematopoietic progenitors belonged to the primitive wave of hematopoiesis. These observations indicate the pres- ence of MYB-dependent primitive erythro-myeloid pro- genitors (EMP) in the early human hematopoietic develop- ment. The issue of definitive versus primitive EMP in humans is disputed;28 it is possible that both primitive and definitive cohorts of EMP participate in the establishment of human hematopoiesis. One report noticed abnormali- ties both in primitive and definitive hematopoiesis in a c-myb mutant of the medaka fish.29 However, the c-myb mutation creates a dominant-negative allele that could inhibit the function of the related a-myb and b-myb genes.30 Other mouse and zebrafish studies unequivocally denied Myb/myb a role in the primitive hematopoiesis.8,22 Our data challenged the notion of MYB as a factor that does not have a function in the primitive hematopoiesis. The differences in the developmental role of MYB between small model animals and humans are likely to be associat- ed with the requirement to facilitate the longer prolifera- tion of primitive blood cells in order to sustain the grow- ing needs of human conceptuses.
An important issue to consider is whether our findings are relevant to the onset of hematopoietic development in the human conceptus. Poor quality and scarcity of 3rd- week human conceptuses prevent reliable analysis of the early human hematopoietic cells.31 Detailed mouse stud- ies, however, suggest that around 300 EryP-CFC on E8.5[1] are unlikely to give rise to about 105 erythroblasts in the E9.5 conceptus,32 and there should be a progenitor-inde- pendent source of the primitive blood cells. Such large
Figure 8. The role of MYB in the human embryonic stem cell-derived hematopoiesis. The MYB gene targeting and the cytokine-free differentiation system revealed progenitor-independent primitive hematopoiesis. Red arrows show MYB-dependent developmental events, black arrows – MYB-independent stages.
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