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Z. Shah et al.
ment. These studies have supported the established notion of MYB as the definitive hematopoietic factor that does not contribute to the development of the primitive wave.9,10
In an attempt to pinpoint the origin of the definitive hematopoiesis, we generated MYB reporter and MYB-null human embryonic stem cells (hESC) lines by gene target- ing and subjected them to hematopoietic differentiation in defined conditions without exogenous hematopoietic cytokines. Unexpectedly, we have found that MYB is essential for the development and proliferation of primi- tive clonogenic progenitors. Our results suggest that the early primitive blood cells can develop independently of the primitive clonogenic progenitors that constitute a sep- arate minor cell population of primitive hematopoiesis.
Methods
The hESC line used in this study was H1 (NIH code WA01). In order to initiate hematopoietic development, briefly formed embryoid bodies (EB) were allowed to attach to surfaces that were coated with extracellular matrix proteins. The cells were differen- tiated in a Stemline®II SFM (Sigma-Aldrich, St. Louis, MO, USA) – based medium supplemented with hVEGF165 (PeproTech, Rocky Hill, NJ). During the first 2 days post-attachment, hBMP4 (PeproTech) was added to initiate mesoderm formation. Additional information on materials and methods is provided in the Online Supplementary Appendix.
Results
Generation of reporter cell lines and bi-allelic inactivation of MYB
In order to create MYB reporter hESC lines, we have introduced alternative fluorescent gene reporters Venus and tdTomato into the second exon of MYB by TALEN- mediated homologous recombination (Figure 1A; Online Supplementary Figure S1A and B). The reporter insert con- taining a strong transcription stop signal has been placed downstream of the transcription elongation attenuation site and the second promoter both located in the first intron.11,12 This position of the reporter genes maximized the probability of making reporter expression closely reflect transcription regulation of MYB.
Properly targeted clones were selected by Southern hybridization with two different biotin-labeled probes (Online Supplementary Figure S1C and D). For bi-allelic inac- tivation, we excised the PGK-PuroR cassette by Cre recom- binase and subjected resulting PuroS clones to the second round of electroporation with the targeting construct and TALEN (Online Supplementary Figure 1E and F). Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blotting showed that the MYB expression in differentiated bi-allelic knockout cells was effectively switched off (Figure 1B and C). Analysis of MYB exonal expression using RNA sequencing of differentiated mutant hESC confirmed that the gene transcription elongation is blocked by the inserted gene cassette (Online Supplementary Figure S2A). The analysis demonstrated that despite the presence of the PGK promoter in the second targeted allele the promoter leakage was negligible. Karyotyping of the targeted cells did not reveal any gross chromosomal aber- rations (Online Supplementary Figure S2B).
MYB is specifically expressed in the early human blood cells
We subjected H1-isogenic hESC reporter lines (single knockout, SKO, cells) and MYB-null lines (double knock- out, DKO, cells) to a modified planar hematopoietic differ- entiation in defined culture conditions.13-15 These condi- tions improve the reproducibility of the data,15 which is critical for reliable phenotypic analysis of the mutant hESC lines. Moreover, we did not add hematopoietic cytokines to the differentiation medium for closer recapit- ulation of the early hematopoietic development. The rationale for adopting such protocol was that high concen- trations of hematopoietic cytokines are unlikely to occur in the early conceptus.16
The recapitulative quality of such hematopoietic cytokine-free in vitro differentiation was manifested by the spontaneous formation of vascular plexus-like structures and transitory blood island-like VE-Cadherin+ cell aggre- gates at which hematopoietic induction occurred in a process similar to the endothelial-to-hematopoietic transi- tion (EHT) (Figure 1D). Single or clumped CD43+ cells emerged on the fringes of the “blood islands” after appar- ent downregulation of VE-Cadherin in the peripheral cells, and the loss of VE-Cadherin was followed by dissociation of the nascent blood cells from the aggregates (Figure 1D). The emerging CD43+ cells induced the expression of key hematopoietic transcription factors, such as GATA1, GATA2, GFI1, GFI1B, KLF1, LMO2, MYB, RUNX1, SPI1, TAL1, which attested the hematopoietic commitment of these cells in contrast to non-hematopoietic CD43-nega- tive cells (Figure 1E). In accordance with the transcrip- tomics data, MYB-Venus+ cells emerge within these in vitro blood islands (Figure 1F). Our observations suggest that the segregation of the early blood cells from the hESC- derived hemogenic endothelium (HE) is similar to the ini- tiation of hematopoiesis in the yolk sac.2
The SKO cells demonstrated vigorous hematopoietic development identical to that of the parent “wild-type” (WT) H1 hESC (Online Supplementary Figure 3A), indicating the absence of non-specific genetic lesions in these cells. Time course quantitation of MYB-Venus fluorescence in SKO cells and MYB mRNA in WT cells showed two peaks of expression, on day 6 and around day 14 (Figure 1G). The close correlation of MYB-Venus flow cytometry of SKO cells and the MYB qRT-PCR data of WT cells indi- cates that the knockin reporter system faithfully reflects MYB expression. Undifferentiated SKO hESC were Venus-negative while the MYB-Venus expression was induced upon the upregulation of the earliest hematopoi- etic markers and concomitant downregulation of pluripo- tency markers (Figure 2A). Starting on day 6 of hematopoietic differentiation, MYB-Venus was specifical- ly expressed in overlapping blood cell populations (Online Supplementary Figure S3A). Within differentiated cultures on day 6 and day 10, the highest level of MYB-Venus expression was observed in the CD41ahighCD235a+ ery- thro-megakaryocyte precursors and the CD34lowCD45+ phenotypic progenitor population, respectively (Online Supplementary Figure S3B and C).
In agreement with the flow cytometry data, RNA sequencing demonstrated around 20 times higher levels of MYB mRNA in day 6 CD43+ cells compared to day 6 CD43- non-blood cells (Figure 2B). The MYB+ /CD43+ cells also selectively expressed high levels of GYPA (CD235a) and ITGA2B (CD41a) mRNA. These markers were shown
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