During adulthood and embryogenesis, fate decisions of hematopoietic stem and progenitor cells (HSPCs), such as specification, self-renewal, and differentiation are tightly regulated by their neighboring niche cells. Moreover, distinct types of niches supply differential cues to direct alternative cell fates for HSPCs. Yet, currently the intrinsic mechanisms balancing HSPC response obliqueness to microenvironmental signals are unknown.
Friend Leukemia integration-1 (Fli-1), is an ETS transcription factor expressed by vascular beds and hematopoietic lineages. Fli-1 belongs to the "heptad factors" which are hypothesized to specify and sustain a hematopoietic cell fate. While Fli-1 overexpression is linked to leukemia, the functional role Fli-1 plays in HSPC specification and maintenance remains undefined.
We show that inducible deletion of Fli-1 using a Rosa-CreERT2 transgenic adult mice (Fli-1ROSAΔ), results in a rapid thrombocytopenia-associated mortality. Transplantation of Fli-1ROSAΔ bone marrow (BM) cells into WT recipients, to exclude vascular-mediated defects, followed by induction of Fli-1 deletion, resulted with the same phenotype. In a set of modulated competitive transplantation experiments (differential induction time points pre- or post-transplant), we observed defective ability of Fli-1ROSAΔ HSPCs to lodge, engraft, and to sustain hematopoiesis post repopulation. Fli-1 deficient HSPCs exhibited reduced quiescent cell cycling status, a hallmark of stemness, and displayed enhanced apoptosis. Thus, Fli-1 is essential for previously unrecognized cell-autonomous HSPC functions.
To determine whether Fli-1 modulates HSPC specification, Fli-1 was conditionally deleted using a developmental VE-cadherin (CDH5)-Cre transgenic model (Fli-1CDH5Δ). This resulted with premature mortality of Fli-1CDH5Δ embryos, accompanied with a hemorrhagic phenotype. Reduced numbers of hematopoietic cells were still detected in the AGM of e10.5 Fli-1CDH5Δ embryos. Conditional Fli-1 deletion using a developmental hematopoietic Vav-1 Cre transgenic model (Fli-1Vav-1Δ) resulted again with premature mortality. Reduced presence of embryonic Fli-1Vav-1Δ liver HSPCs was observed at e12.5. We also applied two in vitro co-culture systems, to study Fli-1 in endothelial to hematopoietic transition (EHT). First, isolated hemogenic endothelial cells (HEC) from WT and Fli-1ROSAΔ embryos were co-cultured with AGM-derived vascular niche. HECs isolated from Fli-1ROSAΔ AGM were still able to convert to CD45+ cells, however these cells did not expand on a vascular niche. Secondly, we have applied an endothelial to hematopoietic reprogramming system in which isolated lung ECs are virally introduced with DOX inducible FosB, Gfi1, Runx1, and Spi1 (FGRS) factors and co-cultured with vascular niche cells. Both WT and Fli-1ROSAΔ ECs were able to acquire a hemogenic like state resulting with a final capacity to convert into hematopoietic cells. Again, Fli-1ROSAΔ cells displayed lesser numbers of CD45+ cells at the end point, presumably due to impaired interaction with the vascular niche. Indeed, reduced expansion capacity was observed both for mature CD45+ and for HSPC derived from Fli-1CDH5Δ AGM region. Adult Fli-1ROSAΔ HSPCs exhibited the same niche-dependent expansion defect. Induction of Fli-1 deletion in vitro in adult HSPCs revealed loss of dependency on vascular niche inductive signals, as no additive expansion effect was observed for Fli-1ROSAΔ HSPCs in the presence of a vascular niche. Hence, Fli-1 is essential for HSPC expansion rather than hematopoietic specification.
Differential RNA-seq analysis combined with epigenetic studies of expanding WT and Fli-1ROSAΔ HSPCs, revealed dysregulation of Fli-1-controlled pathways involved in transduction of microenvironmental signals for self-renewal. Unexpectedly, H3K27Ac analysis, a marker for transcriptional priming, revealed increased global acetylation of Fli-1ROSAΔ HSPCs' chromatin. Therefor, Fli-1 may not only perform as transcription activator, but foremostly as a genomic suppressor via modulation of histone acetylation status.
Decrypting the mechanism(s) by which Fli-1 orchestrates HSPC self-renewal, may promote an improved expansion protocol of human HSPC pre-transplantation, and provide additional insights for microenvironmental sensing by Fli-1-dependent leukemic cells.
Disclosures
No relevant conflicts of interest to declare.
Importance of Matrix-dimensionality in Regulating the Bone Marrow Hematopoietic Cells Pool
