Hematopoietic Support by Endothelium
Authors: Rafii Shahin , Butler Jason M 
Published: October 2019

Abstract
Abstract SCI-45
Accumulating evidence suggest that endothelial cells (ECs) are not simply passive conduits for delivering oxygen and nutrients, but are also endowed with the potential for elaborating potent stem and progenitor cell-active trophogens. These EC-derived trophogens, which we refer to as “angiocrine factors”, are essential for organogenesis, tissue repair as well as tumor growth. Within each organ, specialized ECs establish a unique cellular microenvironment, known as “Vascular Niche”, from which they express membrane-bound or soluble angiocrine factors and deposit extracellular matrix that orchestrate organ regeneration. Sinusoidal ECs (SECs) are prototypical specialized ECs that line the capillaries of certain organs, such as bone marrow, liver and spleen. Sinusoidal ECs could be distinguished from other organ-specific capillaries by the expression of VEGFR3, and lack of expression of Sca1. We have devised physiologically relevant angiogenic models to demonstrate that ECs, through release of angiocrine factors, stimulate long-term in vitro self-renewal of long term-hematopoietic cells (LT-HSCs) and in vivo reconstitution of the LT-HSC pool. In serum/cytokine-free co-cultures supplemented only with the Kit-ligand, ECs stimulated incremental expansion of repopulating CD34−Flt3−cKit+Lineage−Sca1+ LT-HSCs, which retained their self-renewal ability, as determined by single cell and serial transplantation assays. Endothelial cells supported several fold expansion of authentic LT-HSCs beyond 21 days without any evidence of stem cell exhaustion or leukemic transformation. One mechanism by which ECs support LT-HSCs self-renewal is mediated through angiocrine expression of Notch-ligands by ECs, which promote proliferation and prevent exhaustion of LT-HSCs. In support of this notion, we show that endothelial cells supported the expansion of HSCs derived from wild type, but not Notch1/Notch2 deficient mice. Employing the transgenic notch reporter (TNR.Gfp) mice, in which stimulation of the Notch signaling pathway results in GFP expression, we demonstrate that ECs support long-term expansion of TNR.Gfp+cKit+Sca1+Lineage− (TNR.Gfp+KLS), but not Notch1−/−Notch2−/− CD34−Flt-3−KLS LT-HSCs. Remarkably, during hematopoietic recovery from sublethal irradiation, every regenerating TNR.Gfp+cKit+Sca1+Lineage− LT-HSC was detected in close cellular proximity of the SECs. Those TNR.Gfp+cKit+Sca1+Lineage− LT-HSCs that were positioned in the vicinity of the osteoblastic cells were also directly attached to SECs. Notably, selective targeting of the SECs by anti-angiogenic factors, resulted in the downregulation of angiocrine expression of the Notch-ligands resulting in the impaired replenishment of TNR.Gfp+KLS cells. In these experiments the perfusion capacity of the SECs remained intact, suggesting that the angiocrine expression of Notch-ligands by SECs, without contribution from non-EC stromal cells, is sufficient to restore LT-HSC repopulation. Collectively, we demonstrate that within the vascular niche, release of soluble Kit-ligand and angiocrine expression of Notch-ligands by structurally intact SECs establish an instructive niche for the restoration of LT-HCS pool. Endothelial cells provide for an ideal vascular model not only to expand LT-HSCs for therapeutic bone marrow transplantation, but also to identify as yet unrecognized factors that collaborate with Notch and c-Kit signaling to balance LT-HSC expansion and lineage-specific differentiation.
1) Butler JM et al. Instructive role of the vascular niche in promoting tumor growth and tissue repair by angiocrine factors. Nature Reviews Cancer, 2010, 10:138–146.
2) Butler JM et al. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell, 2010, 6, 251–64.
3) Hooper AT et al. Engraftment and reconstitution of hematopoiesis is dependent on VEGFR2-mediated regeneration of sinusoidal endothelial cells. Cell Stem Cell, 2009, 4:263–74.

Disclosures:
No relevant conflicts of interest to declare.