Stem CellsProinflammatory signals are insufficient to drive definitive hematopoietic specification of human HSCs in vitro

- Proinflammatory signaling does not improve definitive hematopoietic stem cell specification in vitro.
- Primitive human pluripotent stem cell hematopoietic derivatives lack an innate proinflammatory phenotype.
- Proinflammatory cytokines do not drive blood formation from human pluripotent stem cells using current protocols.

Recent studies in zebrafish and mice have revealed that proinflammatory signaling is a positive regulator of definitive hematopoietic development. Whether proinflammatory signaling also regulates human hematopoietic specification remains unknown. Here, we explored the impact of the proinflammatory cytokines tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), and interleukin-1β (IL1β) on in vitro hematopoietic differentiation using human pluripotent stem cells. Gene expression analysis and enzyme-linked immunosorbent assay revealed the absence of a proinflammatory signature during hematopoietic development of human pluripotent stem cells. Functionally, the emergence of hemogenic endothelial progenitors (CD31+CD34+CD45− or CD34+CD43−CD73−) and hematopoietic cells (CD43+CD45+) was not affected by treatment with increasing doses of TNFα, IFNγ, and IL1β irrespective of the developmental window or the differentiation protocol used (embryoid body or OP9 co-culture based). Similarly, knockdown of endogenous NF-kB signaling had no impact on hematopoietic differentiation of human pluripotent stem cells. This study serves as a demonstration that TNFα, IFNγ, and IL1β signals do not improve hematopoietic differentiation of human pluripotent stem cells using current protocols and suggests that proinflammatory signaling is insufficient to drive definitive hematopoietic specification of human hematopoietic stem cells in vitro.