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MYB bi-allelic targeting
AB
Figure 4. Innate immunity genes, hematopoietic transcription factors, and cell cycle regulatory genes negatively regulated in MYB-null cells. (A) Heat map of top 20 enriched terms across the two differential gene expression (DEG) lists (upregulated and downregulated, log2FC >2, <−2) in MYB-null cells, colored by P-values. * – R-HSA-210747: “regulation of gene expression in early pancreatic precursor cells”; ** – R-HSA-198933: “immunoregulatory interactions between a lymphoid and a non-lymphoid cell”; *** – GO:0098542: “defense response to other organism”. (B) Bar plots of mRNA expression of cell cycle regulatory genes and hematopoietic transcription factors in wild-type (WT) CD43+, and MYB-Venus+ SKO and DKO cells. Data are mean ± standard error of the mean (SEM), n=3 (biological repeats). P- values were calculated using one-way ANOVA with Tukey's test for multiple comparisons, where ***P<0.001; **P<0.01; *P<0.05.
tures of MYB-null hESC whereas non-macrophage myeloid progenitors are noticeably affected by the MYB inactivation. Such results are unexpected since previous studies denied any role of Myb/myb/MYB in primitive hematopoiesis.7-9, 22 It was reported earlier that BFU-E and CFU-Mix progenitors failed to develop from Myb-null mESC, although whether such progenitors belonged to primitive or definitive hematopoiesis was unclear.23
In order to validate our discovery that MYB was required for the development of primitive human hematopoietic progenitors, we stimulated hESC-derived primitive hematopoiesis Activin A and studied the effects of such treatment on the hematopoietic differentiation of WT, DKO, and SKO cells. Primitive, but not definitive, human hematopoiesis is stimulated by Activin/Nodal sig- naling.17,24 In our experiments, the Activin A treatment led to a reliable increase of MYB-Venus expression in both SKO and DKO blood cells (Figure 5G) and a significant expansion of all types of day 6 clonogenic progenitors derived from the WT hESC (Figure 5H). The latter obser- vation further indicated that all these progenitors were primitive. As expected, MYB-null cells failed to boost the generation of BFU-EP and CFU-MixP progenitors upon the Activin stimulation. The stimulation of CFU-EP was not, however, precluded by the bi-allelic inactivation of MYB, although these MYB-null progenitors formed substandard colonies as described above. Activin-stimulated DKO myeloid progenitors formed only macrophage colonies confirming the resistance of the macrophage lineage to
MYB inactivation.10 These results confirm that MYB is required for the development and/or proliferation of prim- itive human hematopoietic progenitors.
VEGF is a major mitogen of early human hematopoiesis
Our results demonstrated that primitive blood cells emerged normally in the circumstances, at which primi- tive hematopoietic progenitors were severely compro- mised. Apparent progenitor-independence of the early blood cells poses a question of what factors drive the expansion of the early hematopoiesis.
VEGF is the only growth factor that was added to the culture medium after mesoderm induction and, therefore, it is an obvious candidate for a stimulatory molecule pro- moting the emergence and expansion of early blood cells. Indeed, the removal of VEGF from culture medium start- ing from day 3 of the differentiation timecourse led to a strong inhibition, but not a complete suppression, of the early hematopoiesis (Figure 6A), which indicates that this growth factor is responsible for mitogenic support of early human blood cells. In conjunction with the resistance of the early blood cells to MYB inactivation, these observa- tions strongly suggest that MYB and VEGF regulate early human hematopoiesis independently. The VEGF removal also abolished the generation of clonogenic progenitors but did not prevent the robust generation of myeloid cells at the later stages (Figure 6B to D). The progenitor-inde- pendent myeloid cells do not originate from the MYB-
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