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Z. Shah et al.
to be specific for the hESC-derived primitive human hematopoiesis.17,18 Correspondingly, cell populations expressing CD43, CD235a, and CD41a were strongly sup- pressed by SB-431542, the inhibitor of Activin/Nodal sig- naling and primitive human hematopoiesis17 (Figure 2C). Principal component analysis (PCA) revealed a very close similarity of gene expression profiles in day 6 DKO/SKO MYB-Venus+ cells and WT CD43+ primitive blood cells whereas gene expression of day 6 DKO/SKO MYB-Venus– cells closely correlated with the profile of WT CD43– cells (Figure 2D). Taken together, these observations indicate that in contrast to mouse data1,7 MYB is specifically expressed in the primitive human blood cells.
Defective development of myeloid and T cells derived from MYB-null human embryonic stem cells
The MYB-Venus fluorescence in differentiated DKO hESC was much stronger compared to SKO cells, while the hematopoietic specificity of the reporter expression was preserved (Figure 2E). The upregulation of MYB-Venus expression in the MYB-null cells was signifi- cantly higher than the expected two-fold rise in cells with the bi-allelic knockin of a reporter gene (Figure 2F) indicat- ing negative transcriptional autoregulation of MYB tran- scription during the early human hematopoietic develop- ment.
Despite the specific expression of MYB in early human blood cells, its bi-allelic inactivation did not perturb the emergence of blood cells at the early stages of differentia- tion (Figures 2E and 3A). The vast majority of these MYB-independent early blood cell populations were CD235a+/CD41a+ cells of the primitive erythro- megakaryocytic lineage (Figure 3A). Minor qualitative alterations in the expression of hematopoietic markers were noticeable in the CD43+CD45+ DKO cell population starting from day 10 of differentiation. (Figure 3B). The phenotypical differences become increasingly prominent by day 12 (Online Supplementary Figure 4A). By day 20, DKO cells failed to develop into CD14lowCD66b+CD86- neutrophil granulocytes, and the development of CD11b+ immature myeloid cells was also negatively affected (Figure 3C). SKO cells demonstrated an intermediate myeloid phenotype, indicating a dose-dependent manner of MYB contribution to early human myeloid develop- ment and further validating the observed phenotype of MYB-null cells. The emergence of CD14+CD86+ mono- cytes and macrophages was apparently independent of MYB. The defect in the myeloid development of DKO cells was detected already on day 16 (Online Supplementary Figure S4B). Thus, MYB does not participate, or its defi- ciency is compensated, in the initial hematopoietic com- mitment, but the gene is critically important for granulo- cyte development and maturation.
The transcriptomes of MYB-Venus+/CD43+ early blood cell populations sustained significant changes between day 6 and day 12. In contrast, the gene expression profiles of non-blood MYB-Venus-/CD43- remained almost the same (Figure 2D), except day 12 SKO MYB-Venus- popu- lations containing blood cells that by day 12 further decreased the low MYB-Venus fluorescence of SKO cells and were sorted into the MYB-negative population. The day 6 – day 12 shift in gene expression of MYB- Venus+/CD43+ cells reflects a vigorous hematopoietic development in which MYB had limited influence because day 12 DKO/SKO MYB-Venus+ cells continued to cluster
with day 12 WT CD43+ cells. The data support our phe- notypic observations that the functional influence of MYB on the emergence of early blood cells is limited.
Nevertheless, the transcriptomes of DKO/SKO MYB-Venus+ and WT CD43+ cell populations were more divergent by day 12 compared to the close clustering of day 6 populations (Figure 2D). This divergence was suffi- cient for a reliable differential gene expression (DEG) analysis, and the GO term enrichment revealed a clear pattern of expression changes that occurred in MYB-null blood cells. Inactivation of MYB resulted in the downreg- ulation of genes responsible for the innate immune and inflammatory response (Figure 4A; Online Supplementary Figure S5A). In DKO cells, mainly non-hematopoietic genes were upregulated, which suggests that MYB disrup- tion switches hESC differentiation from hematopoietic to non-hematopoietic development. We also found that in addition to known MYB target genes such as GFI1, and BCL2, the MYB deficiency led to the suppression of two key regulators of myeloid differentiation, CEBPA and SPI1 (PU.1) (Figure 4B). Furthermore, the expression of CDK6, a key regulator of HSC activation,19 was inhibited in DKO cells whereas several CDK inhibitors were upregulated including CDKN1A (CIP1/WAF1), a major mediator of p53-dependent tumor suppression.20 The expression of myeloid cytokine receptor genes, CSF1R and CSF3R, as well as a number of genes encoding receptors for pro- and anti-inflammatory cytokines were negatively affected by MYB inactivation (Online Supplementary Figure 5B). The expression of CSF3R was most severely affected, which can explain the observed defects in the granulocyte devel- opment of MYB-null cells. In sum, the transcriptome pro- filing confirmed the involvement of MYB in the develop- ment of innate immune myeloid cells and indicated possi- ble mechanisms of MYB regulation of the early human hematopoiesis.
We also confirmed the crucial role of MYB in the devel- opment of definitive hematopoiesis. Analysis of the T-cell potential is a standard procedure to monitor definitive hematopoietic development in differentiating cultures of hPSC.17 We found that MYB inactivation led to complete failure of lymphoid cell development in the co-culture with OP9-DL4 stroma while WT cells efficiently produced CD5+CD7+ and CD4+CD8+ T cells (Online Supplementary Figure S6).
MYB inactivation results in the deficiency of primitive clonogenic progenitors
Next, we investigated whether MYB is required for the development of clonogenic hematopoietic progenitors. In our differentiation system, the earliest clonogenic progen- itors arise on day 6, and their emergence coincides with the spontaneous formation of the in vitro blood islands (Figure 1D). All erythroid/erythro-myeloid progenitors corresponded to the primitive hematopoietic lineage pro- ducing primitive erythroblasts of characteristic morpholo- gy and primitive megakaryocytes distinguished by their relatively smaller size and less lobular nuclei (Figure 5A).21 Moreover, gene expression profiling of pooled methylcel- lulose colonies showed the overwhelming predominance of embryonic hemoglobin expression (Figure 5B). We des- ignated the hESC-derived primitive erythroid progenitors with higher proliferative potential as BFU-EP (Burst Forming Unit – Erythroid, Primitive), and those with low proliferative potential as CFU-EP (Colony Forming Unit –
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haematologica | 2021; 106(8)