SummaryThe differentiation of human embryonic stem cells (hESCs) to hematopoietic lineages initiates with the specification of hemogenic endothelium, a transient specialized endothelial precursor of all blood cells. This in vitro system provides an invaluable model to dissect the emergence of hematopoiesis in humans. However, the study of hematopoiesis specification is hampered by a lack of consensus in the timing of hemogenic endothelium analysis and the full hematopoietic potential of this population. Here, our data reveal a sharp decline in the hemogenic potential of endothelium populations isolated over the course of hESC differentiation. Furthermore, by tracking the dynamic expression of CD31 and CD235a at the onset of hematopoiesis, we identified three populations of hematopoietic progenitors, representing primitive and definitive subsets that all emerge from the earliest specified hemogenic endothelium. Our data establish that hemogenic endothelium populations endowed with primitive and definitive hematopoietic potential are specified simultaneously from the mesoderm in differentiating hESCs.
Adult-type hematopoietic stem and progenitor cells are formed during ontogeny from a specialized subset of endothelium, termed the hemogenic endothelium, via an endothelial-to-hematopoietic transition (EHT) that occurs in the embryonic aorta and the associated arteries. Despite efforts to generate models, little is known about the mechanisms that drive endothelial cells to the hemogenic fate and about the subsequent molecular control of the EHT. Here, we have designed a stromal line-free controlled culture system utilizing the embryonic pre-somitic mesoderm to obtain large numbers of endothelial cells that subsequently commit into hemogenic endothelium before undergoing EHT. Monitoring the culture for up to 12 days using key molecular markers reveals stepwise commitment into the blood-forming system that is reminiscent of the cellular and molecular changes occurring during hematopoietic development at the level of the aorta. Long-term single-cell imaging allows tracking of the EHT of newly formed blood cells from the layer of hemogenic endothelial cells. By modifying the culture conditions, it is also possible to modulate the endothelial cell commitment or the EHT or to produce smooth muscle cells at the expense of endothelial cells, demonstrating the versatility of the cell culture system. This method will improve our understanding of the precise cellular changes associated with hemogenic endothelium commitment and EHT and, by unfolding these earliest steps of the hematopoietic program, will pave the way for future ex vivo production of blood cells.
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