Angiotensin-Converting Enzyme (ACE) Expression Defines the Earliest Primitive and Definitive Lympho-Hematopoietic Progenitors Derived from Human Embryonic Stem Cells.
Authors: Zambidis Elias T. , Jokubaitis Venta J. , Tam Ada , Sinka Lidia , Civin Curt I. , Tavian Manuela , Simmons Paul J. , Peault Bruno 
Published: October 2019

Abstract
Pluripotent human embryonic stem cells (hESC) provide unprecedented opportunities for studying obscure human developmental events, such as those required for the genesis of hematopoietic stem cells (HSC).
A poorly characterized aspect of embryonic and adult HSC development is the role of the renin-angiotensin system (RAS), which is implicated in regulating HSC proliferation at yolk sac (YS), fetal, and adult stages. We have recently described somatic ACE surface expression not only in adult HSC, but also at the earliest stages of emergent hemato-endotheliogenesis using a novel monoclonal antibody (BB9). ACE expression identifies primitive subsets of adult CD34+ bone marrow HSC, but more intriguingly, marks emergent hematopoietic cells from both CD34− and CD34+ areas of human YS, intraembryonic subaortic patches, and hemogenic endothelial layers of the aorta-gonad-mesonephros (AGM) region. The pattern of human embryonic ACE expression is consistent with the hypothesis of ACE+CD34− hemangioblasts emigrating dorsally from the para-aortic splanchnopleura, and subsequently colonizing the ventral aorta to give rise to CD34+ hemogenic endothelium.
We tested the hypothesis that ACE expression would similarly identify emerging hemato-endothelial progenitors derived from our recently-described hESC-based hematopoietic differentiation system. This embryoid body (hEB)-based system recapitulates hemato-endothelial, primitive, and definitive stages of human embryonic blood development. ACE expression kinetics during hEB differentiation correlated well with the onset of hemato-endothelial differentiation and gene expression (e.g., SCL/tal1, CDX4, CD31, and CD34). Furthermore, using improved, novel methods of hEB hematopoietic differentiation that dramatically augment multilineage progenitors for both primitive and definitive hematopoiesis, we observed that levels of hEB ACE expression were directly correlated to increased hematopoietic potency. Subsequent FACS purification of these hEB cells demonstrated that the earliest detectable multilineage lympho-hematopoietic competency was contained entirely within both ACE+CD34−CD45− hEB and ACE+CD34+CD45− populations. These early ACE+CD45− hEB populations were heterogenous, and co-expressed abundant levels of known hemato-endothelial markers such as CD31, KDR, CD164, CD43, and CD71.
Using novel in vitro assays of primitive and definitive hematopoietic potential, we demonstrated that ACE+ hEB contained common progenitors for both primitive and definitive hematopoiesis, with ACE+CD34−CD45− hEB being enriched for the highest number of progenitors. We were also able to demonstrate that further maturation of these ACE+ hEB cells in an in vitro AGM-like stromal environment produced definitive hematopoietic progenitors that resembled those obtained from cord blood CD34+ cells. The regulatory effects of angiotensin agonist/antagonist peptides on hEB-derived hematopoietic ACE+ progenitors, and their in vivo correlation to ACE+ cells obtained from early human YS and AGM tissue is currently in progress. Furthermore, single-cell analysis is underway to delineate an ACE+ hEB-derived hemangioblastic precursor of not only endothelium, but also primitive and definitive lympho-hematopoiesis.