Human Embryonic Stem Cells Demonstrate That Mpl Expression Initially Appears on a Population of CD31+ Endothelial Cells with Hemogenic Potential.
Authors: Kamata Tamihiro , Gong Yuping , Wang Shaohui , Leavitt Andrew D. 
Published: April 2020

Abstract
Mammalian hematopoiesis occurs adjacent to endothelial cells in the yolk sac and in the AGM region during fetal development, and in vivo cell labeling with Ac-LDL in fetal birds and mice indicates that hematopoietic cells can arise from endothelial cells. Blood forming potential has also been reported in endothelial cells isolated from human fetal liver and fetal bone marrow. However, the role of endothelial cells in the generation of hematopoietic cells remains poorly understood. While thrombopoietin (TPO) and its receptor, Mpl, are known for their critical role in megakaryocytopoiesis, Mpl−/ − mice and patients with congenital amegakaryocytic thrombocytopenia demonstrate that Mpl signaling is also important for the establishment and/or maintenance of hematopoietic stem cells. However, the nature of that role and the relevant Mpl-expressing cells remain unknown. To determine if Mpl signaling in early hematopoiesis involves hemogenic endothelium, we used human embryonic stem cells (hESCs) co-cultured with OP9 stromal cells, a robust model system for generating human hematopoietic cells. We found that CD34+ cells appeared by day 3–4 of co-culture, followed one day later by the appearance of CD31+ cells, nearly all of which appeared within the CD34+ population. The CD34+ cells increased to 20–40% of the total cell population by day 10, with nearly half co-expressing CD31+. Mpl+ cells first appeared on day 6–7, almost exclusively within the CD31+ population, increasing to 5–10% of total cells by day 10. The hESC:OP9 system generated a vascular-like network lined with von Willebrand Factor-expressing cells, which combined with FACS data showing the onset of Mpl expression on CD31+ cells, suggested that Mpl may first be expressed on vascular lining cells. Interestingly, Mpl expression precedes the detection of CD45+ cells, which are first observed on day 8–9. Moreover, round CD45+ cells appeared within the vascular networks, raising the possibility that vascular lining cells may give rise to hematopoietic cells. To begin to characterize the role of TPO/Mpl signaling in the CD31+ cell population, FACS-sorted CD31+ cells from day-9 and day-10 cultures were plated onto a fibronectin (FN)-coated surface, and non-adherent cells were removed after 1 hour. The adherent CD31+ cells uptake Ac-LDL and express vWF, characteristic of endothelial cells. When cultured in media supplemented with endothelial growth factors, including VEGF, EGF, bFGF, IGF-1, and heparin, the FN-adherent cells generated areas of cobblestone-like cell clusters and CD45+/CD34+ cells. When cultured in the same conditions plus TPO, the number of cobblestone-like clusters increased, and the number of CD45+/CD34+ hematopoietic cells generated increased 3–4 fold. Our data demonstrate that Mpl is expressed on a CD31+, vWF-expressing cell population with hemogenic potential, and that TPO/Mpl signaling increases the yield of hematopoietic cells generated from these cells. Ongoing sorting experiments, including the isolation and characterization of CD31+/Mpl+ cells, will further our understanding of where and how Mpl signaling affects early human hematopoietic development. HESCs provide a novel system for defining the role of TPO/Mpl signaling in early human hematopoietic development, which may lead to improved treatment of hematopoietic disorders.