We report that angiotensin-converting enzyme (ACE), a critical physiologic regulator of blood pressure, angiogenesis, and inflammation, is a novel marker for identifying hemangioblasts differentiating from human embryonic stem cells (hESC). We demonstrate that ACE ؉ CD45 ؊ CD34 ؉/؊ hemangioblasts are common yolk sac (YS)-like progenitors for not only endothelium but also both primitive and definitive human lymphohematopoietic cells. Thrombopoietin and basic fibroblast growth factor are identified as critical factors for the proliferation of human hemangioblasts.The developmental sequence of human embryoid body hematopoiesis is remarkably congruent to the timeline of normal human YS development, which occurs during weeks 2 to 6 of human gestation. Furthermore, ACE and the reninangiotensin system (RAS) directly regulate hemangioblast expansion and differentiation via signaling through the angiotensin II receptors AGTR1 and AGTR2. ACE enzymatic activity is required for hemangioblast expansion, and differentiation toward either endothelium or multipotent hematopoietic progenitors is dramatically augmented after manipulation of angiotensin II signaling with either AGTR1-or AGTR2-specific inhibitors. The RAS can therefore be exploited to direct the hematopoietic or endothelial fate of hESC-derived hemangioblasts, thus providing novel opportunities for human tissue engineering. Moreover, the initial events of human hematoendotheliogenesis can be delineated in a manner previously impossible because of inaccessibility to early human embryonic tissues. IntroductionHuman hematopoiesis initiates during the third week of development with formation of yolk sac (YS) blood islands derived from extraembryonic mesoderm. 1,2 The YS generates primarily nucleated primitive erythroblasts that express predominantly embryonic hemoglobins (eg, ε 2 2 , Gower I; ␣ 2 ε 2 , Gower II; and 2 ␥ 2 , Portland), and primitive macrophages. 3 After the onset of circulation at approximately 21 days of embryonic development, YS blood cells continue to circulate in embryonic blood, but the fetal liver (FL) eventually replaces the YS as the main hematopoietic organ beginning at 5 to 6 weeks. FL hematopoiesis is dominated by adult-type definitive erythrocytes (enucleated, macrocytic) expressing abundant fetal (HbF; ␣ 2 ␥ 2 ), adult (HbA; ␣ 2 ␤ 2 ) hemoglobins, but limited embryonic hemoglobins. [4][5][6] The FL also produces rare megakaryocytes, granulocytes, macrophages, lymphocytes, and blast cells. 7 Definitive erythroid, myeloid, megakaryocytic, natural killer-B (NK-B) lymphoid, and multipotent progenitors, with limited long-term engraftment potential, are briefly detected during the late YS (eg, 4-5 weeks) stage, before fetal liver hematopoiesis, in both mice and humans. 2,[8][9] The ephemeral nature of definitivetype YS hematopoiesis suggests a gradual yolk sac-fetal liver transition. 1 Indeed, the classic embryonic 3 fetal 3 adult globin switch in early erythrocytes occurs sequentially over time and probably in the same clonal lineage. For examp...

Context Bilateral adrenal hemorrhage is a rare condition with potentially life-threatening consequences as acute adrenal insufficiency. Early adrenal axis testing, as well as directed imaging, is crucial for immediate diagnosis and treatment. Coronavirus disease 2019 (COVID-19) has been associated with coagulopathy and thromboembolic events. Case decription A 66-years-old woman presented with acute COVID-19 infection and primary adrenal insufficiency due to bilateral adrenal hemorrhage (BAH). She had also a renal vein thrombosis. Her past medical history revealed primary antiphospholipid syndrome (APLS). 4 weeks after discharge she had no signs of COVID-19 infection and her PCR test for COVID-19 was negative, but she still needed glucocorticoid and mineralocorticoid replacement therapy. The combination of APLS and COVID-19 was probably responsible of the adrenal event as a "two-hit" mechanism. Conclusions COVID-19 infection is associated with coagulopathy and thromboembolic events, including BAH. Adrenal insufficiency is life threatening, therefore we suggest to consider performing early adrenal axis testing for COVID-19 patients with clinical suspicion of adrenal insufficiency.

Recent characterization of hemangioblasts differentiated from human embryonic stem cells (hESC) has further confirmed evidence from murine, zebrafish and avian experimental systems that hematopoietic and endothelial lineages arise from a common progenitor. Such progenitors may provide a valuable resource for delineating the initial developmental steps of human hemato-endotheliogenesis, which is a process normally difficult to study due to the very limited accessibility of early human embryonic/fetal tissues. Moreover, efficient hemangioblast and hematopoietic stem cell (HSC) generation from patient-specific pluripotent stem cells has enormous potential for regenerative medicine, since it could lead to strategies for treating a multitude of hematologic and vascular disorders. However, significant scientific challenges remain in achieving these goals, and the generation of transplantable hemangioblasts and HSC derived from hESC currently remains elusive. Our previous work has suggested that the failure to derive engraftable HSC from hESC is due to the fact that current methodologies for differentiating hESC produce hematopoietic progenitors developmentally similar to those found in the human yolk sac, and are therefore too immature to provide adult-type hematopoietic reconstitution. Herein, we outline the nature of this challenge and propose targeted strategies for generating engraftable human pluripotent stem cell-derived HSC from primitive hemangioblasts using a developmental approach. We also focus on methods by which reprogrammed somatic cells could be used to derive autologous pluripotent stem cells, which in turn could provide unlimited sources of patient-specific hemangioblasts and HSC.

We have defined serum-free (SF) culture conditions for efficiently generating clonogenic hemangioblasts from pluripotent human embryonic stem cells (hESC). We also report that angiotensin-converting enzyme (ACE), a critical physiologic regulator of blood pressure, angiogenesis, and inflammation, is a novel marker for identifying and purifying hemangioblastic stem-progenitors from differentiating hESC. Using in vitro clonal assays of primitive and definitive hematopoietic potential, we demonstrated that ACE+ human embryoid body (hEB) cell populations contain a common yolk sac (YS)-like progenitor for not only endothelium, but also both primitive and definitive hematopoieses. Thrombopoietin (TPO) and basic fibroblast growth factor (FGF2) were identified as critical growth factors for expanding ACE+ hemangioblasts. ACE+CD34-CD45- human embryoid body (hEB) cells contained the highest number of these progenitors. Further maturation of ACE+ hemangioblasts in a stromal environment produced definitive-type lympho-hematopoietic progenitors, albeit with a late YS, and not AGM phenotype. The developmental kinetics of YS-like hematopoiesis generated from hEB cells was remarkably in parallel to the actual timeline of human YS development, which occurs during weeks 2–6 of human gestation. We also showed that ACE and the renin-angiotensin system (RAS) axis plays a direct role in hESC-derived hemangioblastic differentiation, and is directly regulated via signaling through the two major angiotensin peptide receptors (AGTR1 and AGTR2). Moreover, directed differentiation of hemangioblasts toward either endothelium, or multipotent primitive hematopoietic progenitors was efficiently enhanced via modulation of either AGTR1 or AGTR2 signaling. RAS axis manipulation may therefore be exploited for directing the differentiation of transplantable hESC-derived hemangioblastic stem-progenitors, thus providing novel opportunities for human tissue engineering. The key molecular and cellular events of human hemato-endothelial genesis can now be delineated in a manner that was previously impossible due to inaccessibility of human embryonic tissue. Figure Figure