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Z. Shah et al.
was at a very low but detectable level at both stages. These results demonstrated that the gene is required not only for progenitor proliferation but also for their develop- ment from differentiated hESC. In addition, the progenitor recovery further evidenced that defects in the develop- ment of primitive hematopoietic progenitors were associ- ated with the MYB inactivation rather than nonspecific genomic abnormalities.
We also studied whether the reactivation of MYB in the MYB-null cells could rescue maturating myeloid cells at day 20 of the primary differentiation. As shown in Figure 7E, the continuous expression of PB-MYB from day 4 to day 18 has led to a robust recovery of CD11b+ and CD11c+ monocyte-macrophage cells with strong upregu- lation of CD86 and HLA-DR. In contrast, only limited recovery of CD66b+ granulocytes was observed on day
ABC
D
E
Figure 7. MYB is required for both emergence and proliferation of the primitive clonogenic progenitors. (A) Scheme of the PiggyBac-MYB transposon construct. (B) Western blotting of doxyclycline (DOX)-induced expression of transgenic MYB in day 12 DKO1-PB-MYB and DKO1-PB-MYB-Cherry cells. (C) A representative CFU-mix colony generated by DOX-stimulated day 6 DKO1-PB-MYB-Cherry cells. Scale bar, 100 mm. (D) Efficient rescue of day 6 clonogenic progenitors is achieved when DOX is added to both primary and secondary stages of hematopoietic differentiation of DKO1-PB-MYB cells. Data are mean ± standard deviation (SD), n=3. (E) MYB over- expression in the DKO1-PB-MYB clones during day 4 to day 18 period leads to the accumulation of immature myeloid cells on day 20. Representative data of three experiments are shown.
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haematologica | 2021; 106(8)