This study was aimed to investigate whether endothelium-specific deletion of PTEN can affect hemangioblast development in the AGM region of mouse embryos. Based on Cre/loxP system, the Tie2CrePten(loxp/loxp) and Tie2CrePten(loxp/wt) mouse embryos were obtained. The genotype was identified by PCR. After treated with type I collagenase, the AGM region was dispersed into single-cell suspension, and then was cultured in blast colony-forming cell (BL-CFC) media. The number of BL-CFC was counted 4 or 5 days later. The hematopoietic capacity of BL-CFC was detected in methylcellulose culture system and the endothelial potential was assessed by tube-like structure formation on Matrigel. The results showed that the number of BL-CFC in AGM region of Tie2CrePten(loxp/loxp) mouse embryo decreased as compared with Tie2CrePten(loxp/wt) embryo. Whereas the hematopoietic capacity of mutant BL-CFC was enhanced, the endothelial potential, as evaluated by tube-like structure formation in vitro, was significantly reduced. It is concluded that the endothelial PTEN is capable of exerting regulatory functions on both the numbers and the dual potential of hemangioblast in mouse AGM region.
Animals ; Cell Differentiation ; Cells, Cultured ; Hemangioblasts ; Hematopoietic Stem Cells ; cytology ; Mice ; PTEN Phosphohydrolase ; genetics

OBJECTIVETo explore the role of bromodomain and extra terminal (BET) bromodomain in hematopoietic differentiation from human enbryonic stem cells (hESC).

METHODSThe effect of BET hematopoietic inhibitor I-BET151 on hematopoietic differentiation from hESC was detected by using a monolayer hematopoietic defferentiation model, immunofluorescence, flow cytometry and real-time PCR; moreover the role of I-BET151 in process of hematopoietic differentiation was explored by adding I-BET151 in different differentiation stages.

RESULTSThe analysis results of immunofluorescence, flow cytometry and real-time PCR showed that I-BET 151 significantly inhibited the generation of CD43 positive hematopoietic stem and progenitor cells (HSPCs). It was found that the addition of I-BET 151 in different stages, including APLNR lateral plate mesoderm production, CD34CD31 hemogenic endothelium (HEP) generation and endothelial-to-hematopoietic transition, significantly suppressed the generation of CD43 positive hematopoietic progenitor cells.

CONCLUSIONI-BET 151 inhibites hematopoietic differentiation from hESCs at several stages, suggesting that the BET bromodomain plays important roles in multiple stages of hematopoietic differentiation from hESCs.

Apelin Receptors ; Cell Differentiation ; Flow Cytometry ; Hemangioblasts ; Hematopoietic Stem Cells ; Human Embryonic Stem Cells ; Humans

Hemangioblasts or blood islands only arise in early development thereby the sources to obtain these bi-potential cells are limited. While previous studies have isolated both lineages in vitro through the hemangioblast, derivation efficiency was rather low due to cellular damage attributed by enzyme usage and fluorescent activated cell sorting (FACS). This study focused on avoiding the use of damaging factors in the derivation of endothelial cells (ECs). Single cell H9-human embryonic stem cells (hESCs) were obtained by using a mild dissociation protocol then human embryoid body (hEB) formation was performed under hemangioblast differentiation conditions. The hEBs were subjected to a two-stage cytokine treatment procedure. Subsequent culture of the adhesive cells in day 4 hEBs gave arise to a seemingly pure population of ECs. The hESC-derived ECs were characterized by identifying signature endothelial gene and protein markers as well as testing for in vitro functionality. Furthermore, in vivo functionality was also confirmed by transplanting the cells in hindlimb ischemic murine models. We demonstrate that the genetic change required for EC derivation precedes blast colony formation. Furthermore, cell damage was prevented by abating enzyme usage and FACS, resulting in a high yield of ECs upon adhesion. Under this method, confluent cultures of ECs were obtainable 4 days after hEB formation which is significantly faster than previous protocols.
Adhesives ; Animals ; Embryoid Bodies ; Embryonic Stem Cells ; Endothelial Cells* ; Hemangioblasts ; Hindlimb ; Human Embryonic Stem Cells* ; Humans* ; In Vitro Techniques ; Islands ; Methods

To provide a guideline of accurate diagnosis and proper methods of treatment of meningioma, the most common benign intracranial tumor, the authors studied 94 cases of intra & extracranial meningioma who had been diagnosed and operated at the Department of Neurosugery, Yonsei University College of Medicine from November, 1966 to October, 1982. The results were as followings. 1) The ratio of male to female was 1 to 1.4 and 30.8% of the 94 patients were in the 4th decade of age and another 30.8% in the 5th decade. 2) The predilection sites were parasagittal and falx(37.2%), sphenoid ridge(15.9%), convexity(14.9%), and suprasellar(10.6%) area. 3) The common initial clinical features were headache(55.5%), visual disturbance (16.0%) and seizure (14.9%). In radiologic diagnostic tests, there were abnormal findings in over a half on plain skull X-ray, and brain CT scan was regarded as the most accurate and safe method. Cerebral angiography provided the informations such as vascularity, venous drainage and relationship with the venous sinus. 4) Among the 6 cases of basal posterior fossa meningioma, the surgical excision of the tumor could be performed totally in 2 cases and subtotally in 4. 5) In 8 recurrent cases, all the tumors except one were located at parasagittal area and they were 3 hemangiopericytic, 2 hemangioblastic, 2 angiomatous and 1 meningotheliomatous type. 6) The Laser was thought to be very effective surgical tool to remove vascular tumor, such as meningioma, with its unique properties of non-mechanical bloodless evaporation of tumor and minimizing of brain edema.
Brain ; Brain Edema ; Cerebral Angiography ; Diagnosis ; Diagnostic Tests, Routine ; Drainage ; Female ; Hemangioblasts ; Humans ; Male ; Meningioma* ; Recurrence ; Seizures ; Skull ; Tomography, X-Ray Computed

In contrast to primitive hematopoiesis, during embryonic definitive hematopoiesis, it has been demonstrated that multilineage hematopoietic stem/progenitor cells (HSPCs) arise from hemogenic endothelium, and the endothelial to hematopoietic transition (EHT) exists within the yolk sac, placenta, AGM, mouse head vascular and extraembryonic vessels. However, whether hemogenic endothelial cells contribute to blood cell development at other sites of definitive hematopoiesis, including fetal liver and bone marrow, remains largely unknown. Recently, more and more researches showed that hematopoiesis within bone marrow had a close relationship with vascular endothelium development, too. This review summarizes the mechanism of EHT during embryo development, and discuss whether EHT exists in adult hematopoiesis.
Animals ; Bone Marrow ; Cell Differentiation ; Embryonic Development ; Endothelial Cells ; Endothelium ; Endothelium, Vascular ; Female ; Hemangioblasts ; Hematopoiesis ; Hematopoietic Stem Cells ; Mice ; Placenta ; Pregnancy ; Yolk Sac

OBJECTIVETo explore the role of sonic hedgehog (Shh) pathway in regulating the proliferation, migration and differentiation of hemangioblasts derived from aorta-gonad-mesonephros (AGM).

METHODSThe hemangioblasts were isolated from AGM region of 11-day postcoitum (dpc) murine embryos by using the immuno-magnetic with CD34 and Flk1 monoclonal antibodies. The phenotypic analysis of hemangioblasts and AGM-derived stromal cells were detected by flow cytometry. The secretion of Shh was examined by immunohistochemical staining. The roles of Shh in regulating the proliferation, migration and differentiation of hemangioblasts in the transwell non-contact coculture system with AGM-derived stromal cells were observed by adding exogenous Shh N-Terminus and its antibody.

RESULTSThe protein of Shh was highly expressed on AGM-derived stromal cells. The proliferation of hemangioblasts was promoted when co-cultured with AGM-derived stromal cells, and the effects of the latter could be blocked by antibody of Shh. The proliferation of hemangioblasts was strengthened further and kept for a long time without differentiation and apoptosis when exogenous Shh N-Terminus was added into the transwell non-contact co-culture system with AGM-derived stromal cells. When exogenous Shh N-Terminus was added into the cultural supernatant of hemangioblasts without AGM-derived stromal cells, the hemangioblasts were observed to be induced to apoptosis or differentiation after a short time of proliferation. Furthermore, the ability of migration could be promoted in the co-cultured hemangioblasts by adding exogenous Shh N-Terminus.

CONCLUSIONShh pathway probably involves in the regulation of the proliferation, differentiation, apoptosis and migration of hemangioblasts, and is regulated by the AGM microenvironment.

Adrenal Glands ; cytology ; Animals ; Aorta ; cytology ; Cell Differentiation ; Cell Movement ; Cell Proliferation ; Cells, Cultured ; Embryo, Mammalian ; cytology ; Hedgehog Proteins ; metabolism ; Hemangioblasts ; metabolism ; physiology ; Mesonephros ; cytology ; Mice ; Mice, Inbred BALB C ; Signal Transduction