In Vitro Generation of Lymphohematopoietic Cells from Endothelial Cells Purified from Murine Embryos

Abstract

We have investigated the lymphohematopoietic potentials of endothelial cells (EC) and hematopoietic cells (HPC) sorted from embryos. Expression of VE-cadherin, CD45, and Ter119 was used to distinguish EC (VE-cadherin+CD45-Ter119-) from HPC (VE-cadherin-CD45+). Thus defined, EC population takes up acetylated LDL and coexpresses CD31, Flk1, and CD34. In E9.5 embryos, EC from yolk sac (YS) and the embryo proper generate blood cells, including lymphocytes. Thus, lymphohematopoietic EC do exist in the embryo, and they are generated both in YS and the embryo proper. On the other hand, HPC with lymphopoietic potency appear first in the embryo proper. These findings implicate involvement of multiple environmental cues for acquiring lymphopoietic competency during differentiation of HPC.

A short history of hemogenic endothelium

“…However, based on expression of the transcription factor Runx1 (discussed below) only a subset of the endothelium in these sites is believed to be hemogenic [36]. Functionally, hemogenic endothelium has been defined as displaying an endothelial phenotype and morphology and having the capacity to form hematopoietic offspring and endothelial tubules/sheets in culture [37,38]. The overlap in cell surface marker expression on (hemogenic) endothelial and hematopoietic cells has further confounded the identification and isolation of hemogenic endothelium.…”

“…Combination of VECadh or CD31 with the hematopoietic markers CD41 [40][41][42][43], CD45 [44] and Ter119 [45] has been used to separate endothelial from hematopoietic cells (e.g. [38,[46][47][48]), and other identification strategies have also been employed e.g. [49] [50].…”

Blood-Forming Endothelium in Human Ontogeny: Lessons From In Utero Development and Embryonic Stem Cell Culture

“…Intraembryonic HSCs were noted to arise on the luminal aspect of endothelial cells in the dorsal aorta and vitelline artery. Diverse lineagemarking experiments based on metabolic labeling of endothelium in avian embryos in ovo (Jaffredo et al 1998), purification of mouse embryonic endothelium by flow cytometry (Nishikawa et al 1998), or tagging of angio-hematopoietic cells in transgenic mice (North et al 2002) have all suggested the conclusion that discrete subsets of vascular endothelial cells transiently exhibit blood-forming potential during vertebrate development, as originally suggested by Sabin (1920).…”

“…It should be kept in mind that hematopoiesis from vessel walls was likely an ancestral mechanism of blood cell generation in evolution, inasmuch as, in addition to converging studies in bird and mouse embryos (Jaffredo et al 1998, Nishikawa et al 1998, North et al 2002, Bollerot et al 2005, similar hematogenous embryonic aortic cells have been characterized in amphibian and fish embryos (Ciau-Uitz et al 2000, Gering andPatient, 2005). Important genetic analyses performed in the same lower vertebrates have confirmed that molecular signaling through Hedgehog, Notch, and VEGF is essential to the development of both adult hematopoietic cells and the dorsal aorta, further associating the emergence of HSCs with the aortic blood-forming endothelium (Brown et al 2000, Gering andPatient, 2005).…”

“…Sabin (1920) first proposed the hypothesis that blood cells can sprout from vascular endothelium, based on microscopic observations of the live chicken yolk sac. These observations fell into oblivion until the end of the century, when several investigators used cell marking, cell sorting, genetic and transgenic techniques to document the existence of blood-forming endothelial cells in the amphibian, fish, avian, and mammalian embryonic aorta (Jaffredo et al 1998, Nishikawa et al 1998, Ciau-Uitz et al 2000, North et al 2002, Oberlin et al 2002, Bollerot et al 2005, Gering and Patient, 2005. Recent availability of first-month human embryos to experimentation, as well as the development of reliable and sensitive assays for human angiogenesis and lymphohematopoiesis, has permitted the precise analysis of incipient angio-hematopoiesis in human ontogeny (Figure 1).…”