scholarly journals Embryonic stem cell-derived cystic embryoid bodies form vascular channels: an in vitro model of blood vessel development

Development ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 303-316 ◽  
Author(s):  
R. Wang ◽  
R. Clark ◽  
V.L. Bautch
Keyword(s):  
Endothelial Cells ◽  
Blood Vessel ◽  
Light And Electron Microscopy ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Density Lipoprotein ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Vascular Channels

Murine embryonic stem cells can differentiate in vitro to form cystic embryoid bodies (CEB) that contain different structures and cell types. The blood islands are one such structure that consist of immature hematopoietic cells surrounded by endothelial cells, the first identifiable vascular cells. CEBs differentiated in vitro developed blood islands initially, and subsequently these blood islands matured to form vascular channels containing hematopoietic cells. Phase contrast microscopy demonstrated the presence of channels in mature CEBs grown in suspension culture, and high resolution light and electron microscopy showed that the cells lining these channels were endothelial cells. The channels appeared less organized than the vasculature of the mature yolk sac. The hematopoietic cells were occasionally seen ‘flowing’ through the CEB channels, although their numbers were reduced relative to the yolk sac. Analysis of primary CEB cultures showed the presence of cells with two characteristics of endothelial cells: approximately 30% of the cells labelled with fluorescent acetylated low density lipoprotein and a small number of cells were positive for von Willebrand's factor by immunostaining. Thus we conclude that a primitive vasculature forms in CEBs differentiated in vitro, and that not only primary differentiation of endothelial cells but also some aspects of vascular maturation are intrinsic to this cell culture system. CEBs are therefore a useful model for the study of developmental blood vessel formation.

Endothelial Differentiation of Human Embryonic Stem Cell and Functional Blood Vessels Formation in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5455-5455
Author(s):  
Zongjin Li ◽  
Bryan Smith ◽  
Mei Huang ◽  
Xiaoyan Xie ◽  
Sanjiv Sam Gambhir ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Imaging Techniques ◽  
Embryonic Stem ◽  
Density Lipoprotein ◽  
Embryoid Bodies ◽  
Endothelial Differentiation ◽  
Differentiation Efficiency

Abstract Background: Human embryonic stem (hES) cells are distinguished by their capacity for self-renewal and pluripotency. Here we characterize the differentiation of hES cell-derived endothelial cells (hESC-ECs), use molecular imaging techniques to examine their survival and function in vivo. Methods and Results: Here we introduced two-step procedures to increase endothelial differentiation efficiency of hESCs by subcultured embryoid bodies (EBs) in collagen. Single cell suspensions from EBs sprouting in collagen were obtained by treatment with collagenase and Liberase Blendzyme IV and CD31/CD144 positive cells were isolated by FACScan. After isolation, these hESC-ECs express endothelial cell markers similar to HUVEC, form vascular-like channels, and incorporate DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) in vitro. Real time PCR array described increasing endothelial transcription. Using whole genome microarrays, we investigated the hESCs derived endothelial cells (hESC-ECs) transcriptome that occur among sequenced hESCs differentiation processes and human umbilical vein endothelial cells (HUVECs). We found that hESC-ECs expressed endothelial gene at pattern similar to HUVECs. By intravital microscope, we demonstrated that hESC-ECs can form function vessels with blood flow. Conclusion: Taken together, two-steps procedures increased the endothelial differentiation efficiency hESCs, and hESC-ECs can form functional vessel in vivo.

Hedgehog is required for murine yolk sac angiogenesis

Development ◽  
2002 ◽  
Vol 129 (2) ◽  
pp. 361-372 ◽  
Author(s):  
Noah Byrd ◽  
Sandy Becker ◽  
Peter Maye ◽  
Roopa Narasimhaiah ◽  
Benoit St-Jacques ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Vessels ◽  
Vascular Remodeling ◽  
Hedgehog Signaling ◽  
Es Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Visceral Endoderm

Blood islands, the precursors of yolk sac blood vessels, contain primitive erythrocytes surrounded by a layer of endothelial cells. These structures differentiate from extra-embryonic mesodermal cells that underlie the visceral endoderm. Our previous studies have shown that Indian hedgehog (Ihh) is expressed in the visceral endoderm both in the visceral yolk sac in vivo and in embryonic stem (ES) cell-derived embryoid bodies. Differentiating embryoid bodies form blood islands, providing an in vitro model for studying vasculogenesis and hematopoiesis. A role for Ihh in yolk sac function is suggested by the observation that roughly 50% of Ihh–/– mice die at mid-gestation, potentially owing to vascular defects in the yolk sac. To address the nature of the possible vascular defects, we have examined the ability of ES cells deficient for Ihh or smoothened (Smo), which encodes a receptor component essential for all hedgehog signaling, to form blood islands in vitro. Embryoid bodies derived from these cell lines are unable to form blood islands, and express reduced levels of both PECAM1, an endothelial cell marker, and α-SMA, a vascular smooth muscle marker. RT-PCR analysis in the Ihh–/– lines shows a substantial decrease in the expression of Flk1 and Tal1, markers for the hemangioblast, the precursor of both blood and endothelial cells, as well as Flt1, an angiogenesis marker. To extend these observations, we have examined the phenotypes of embryo yolk sacs deficient for Ihh or Smo. Whereas Ihh–/– yolk sacs can form blood vessels, the vessels are fewer in number and smaller, perhaps owing to their inability to undergo vascular remodeling. Smo–/– yolk sacs arrest at an earlier stage: the endothelial tubes are packed with hematopoietic cells, and fail to undergo even the limited vascular remodeling observed in the Ihh–/– yolk sacs. Our study supports a role for hedgehog signaling in yolk sac angiogenesis.

scholarly journals Erythropoiesis and vasculogenesis in embryoid bodies lacking visceral yolk sac endoderm

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3720-3730 ◽  
Author(s):  
M Bielinska ◽  
N Narita ◽  
M Heikinheimo ◽  
SB Porter ◽  
DB Wilson
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Visceral Endoderm ◽  
Cell Stage ◽  
Primitive Hematopoiesis

During mouse embryogenesis the first hematopoietic and endothelial cells form in blood islands located between layers of visceral endoderm and mesoderm in the yolk sac. The role of visceral endoderm in primitive hematopoiesis and vasculogenesis is not well understood. We have assessed the consequences of a lack of visceral endoderm on blood cell and vessel formation using embryoid bodies derived from mouse embryonic stem (ES) cells deficient in GATA-4, a transcription factor expressed in yolk sac endoderm. When differentiated in vitro, these mutant embryoid bodies do not develop an external visceral endoderm layer. We found that Gata4-/-embryoid bodies, grown either in suspension culture or attached to a substratum, are defective in primitive hematopoiesis and vasculogenesis as evidenced by a lack of recognizable blood islands and vascular channels and a reduction in the expression of the primitive erythrocyte marker epsilon y-globin. Expression of the endothelial cell transcripts FIk-1, FIt-1, and platelet-endothelial cell adhesion molecule (PECAM) was not affected in the mutant embryoid bodies. Gata4-/-ES cells retained the capacity to differentiate into primitive erythroblasts and endothelial cells when cultured in methylcellulose or matrigel. Analysis of chimeric mice, generated by injecting Gata4-/-ES cells into 8-cell stage embryos of ROSA26 transgenic animals, showed that Gata4-/-ES cells can form blood islands and vessels when juxtaposed to visceral endoderm in vivo. We conclude that the visceral endoderm is not essential for the differentiation of primitive erythrocytes or endothelial cells, but this cell layer plays an important role in the formation and organization of yolk sac blood islands and vessels.

Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies

Development ◽  
1988 ◽  
Vol 102 (3) ◽  
pp. 471-478 ◽  
Author(s):  
W. Risau ◽  
H. Sariola ◽  
H.G. Zerwes ◽  
J. Sasse ◽  
P. Ekblom ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Blood Vessels ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Activity Increase ◽  
Vasculogenesis And Angiogenesis

Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture, they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands, a process which we call vasculogenesis, was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum, fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response, which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, and angiogenesis, the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated.

Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2740-2749 ◽  
Author(s):  
CD Helgason ◽  
G Sauvageau ◽  
HJ Lawrence ◽  
C Largman ◽  
RK Humphries
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Homeobox Genes ◽  
Embryoid Bodies ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Differentiation And Proliferation

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

Hematopoietic Differentiation of Human Embryonic Stem Cells in Vitro : Comparison of Three Cell Lines

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4787-4787
Author(s):  
Marion Brenot ◽  
Annelise Bennaceur-Griscelli ◽  
Marc Peschanski ◽  
Maria Teresa Mitjavila-Garcia
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Human Embryonic Stem Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem

Abstract Human embryonic stem cells (hES) isolated from the inner cell mass of a blastocyst have the ability to self renew indefinitely while maintaining their pluripotency to differentiate into multiple cell lineages. Therefore, hES represent an important source of cells for perspective cell therapies and serve as an essential tool for fundamental research, specifically for understanding pathophysiological mechanisms of human diseases for the development of novel pharmacological drugs. The generation of hematopoietic stem cells from hES may serve as an alternative source of cells for hematopoietic reconstitution following bone marrow transplantation and an interesting approach to understand early stages of hematopoietic development which are difficult to study in human embryos. Using two different methods, we have differentiated three hES cell lines (SA01, H1 and H9) into hematopoietic cells by generating embryoid bodies and co-culturing on the murine Op9 cell line. In both experimental approaches, we obtain cells expressing CD34 and when cultured in hematopoietic conditions, SA01 and H1 cell lines differentiate into various hematopoietic lineages as demonstrated by BFU-E, CFU-GM and CFU-GEMM colony formation, whereas H9 have almost exclusively granulo-macrophage differentiation. Cells composing these hematopoietic colonies express CD45, CD11b, CD31, CD41 and CD235 and staining with May Grundwald-Giemsa demonstrate neutrophil and erythrocyte morphology. These results demonstrate the capacity of hES to differentiate into mature hematopoietic cells in vitro. Nevertheless, there exist some quantitative and qualitative differences about hematopoietic differentiation between the hES cell lines used. However, we still have to evaluate their capacity to reconstitute hematopoiesis in vivo in an immune deficient mouse model. We will also be interested in developing in vitro methods to expand these hematopoietic precursor cells derived from hES which may be used as a viable source for future cell therapy.

Expression of CD41 on hematopoietic progenitors derived from embryonic hematopoietic cells

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 2003-2013 ◽  
Author(s):  
Maria Teresa Mitjavila-Garcia ◽  
Michel Cailleret ◽  
Isabelle Godin ◽  
Maria Manuela Nogueira ◽  
Karine Cohen-Solal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Progenitor

In this study, we have characterized the early steps of hematopoiesis during embryonic stem cell differentiation. The immunophenotype of hematopoietic progenitor cells derived from murine embryonic stem cells was determined using a panel of monoclonal antibodies specific for hematopoietic differentiation antigens. Surprisingly, the CD41 antigen (αIIb integrin, platelet GPIIb), essentially considered to be restricted to megakaryocytes, was found on a large proportion of cells within embryoid bodies although very few megakaryocytes were detected. In clonogenic assays, more than 80% of all progenitors (megakaryocytic, granulo-macrophagic, erythroid and pluripotent) derived from embryoid bodies expressed the CD41 antigen. CD41 was the most reliable marker of early steps of hematopoiesis. However, CD41 remained a differentiation marker because some CD41– cells from embryoid bodies converted to CD41+ hematopoietic progenitors, whereas the inverse switch was not observed. Immunoprecipitation and western blot analysis confirmed that CD41 was present in cells from embryoid bodies associated with CD61 (β3 integrin, platelet GPIIIa) in a complex. Analysis of CD41 expression during ontogeny revealed that most yolk sac and aorta-gonad-mesonephros hematopoietic progenitor cells were also CD41+, whereas only a minority of bone marrow and fetal liver hematopoietic progenitors expressed this antigen. Differences in CD34 expression were also observed: hematopoietic progenitor cells from embryoid bodies, yolk sac and aorta-gonad-mesonephros displayed variable levels of CD34, whereas more than 90% of fetal liver and bone marrow progenitor cells were CD34+. Thus, these results demonstrate that expression of CD41 is associated with early stages of hematopoiesis and is highly regulated during hematopoietic development. Further studies concerning the adhesive properties of hematopoietic cells are required to assess the biological significance of these developmental changes.

α4-Integrin+ Endothelium Derived from Primate Embryonic Stem Cells Generates Both Primitive and Definitive Hematopoietic Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 683-683
Author(s):  
Gen Shinoda ◽  
Katsutsugu Umeda ◽  
Toshio Heike ◽  
Masato Arai ◽  
Akira Niwa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Endothelial Cells ◽  
Embryonic Stem Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Almost All ◽  
Definitive Erythropoiesis ◽  
Definitive Hematopoiesis

Abstract The mechanism of commencement of hematopoiesis in blood islands of the yolk sac and the aorta-gonad-mesonephros (AGM) region during primate embryogenesis remains elusive. We previously showed the development of both primitive and definitive hematopoiesis when cynomolgus monkey embryonic stem cells were co-cultured with OP9 stromal cells. In this study, we examined the hematopoietic potential of endothelial cells developing in our coculture system and demonstrated that VE-cadherin+CD45− endothelial cells derived from embryonic stem cells were able to generate primitive and definitive hematopoietic cells sequentially, as revealed by immunostaining of floating erythrocytes and colony-forming assay in cultures. All floating erythrocytes which emerged initially expressed ε- and ζ-globins, while β-globin expression was hardly detected. The percentage of floating erythrocytes positive for β-globin gradually increased thereafter, and almost all erythrocytes were positive by day 40. Meanwhile, expression of ε- and ζ-globins declined gradually. Clonal analysis revealed that single bipotential cells for hematopoietic and endothelial lineages were included in this endothelial cell population. Hemogenic activity of endothelial cells was observed exclusively in the α4-integrin+ subpopulation. RT-PCR data showed that Runx1, a transcriptional factor associated with definitive hematopoiesis, was expressed in the hemogenic α4-integrin+ subpopulation, but not the non-hemogenic α4-integrin− subpopulation among embryonic stem cell-derived endothelial cells. The kinetics of this hemogenic subpopulation was similar to that of hemogenic endothelial cells previously reported in the yolk sac and the AGM region in vivo, in that they emerged only for a limited time. On the other hand, VE-cadherin−CD45−α4-integrin+ cells gave rise to more primitive erythrocytes than VE-cadherin+CD45−α4-integrin+ cells, but hardly contributed to definitive hematopoiesis. These results indicate that VE-cadherin+CD45−α4-integrin+ endothelial cells generate primitive and definitive hematopoietic cells sequentially, while VE-cadherin−CD45−α4-integrin+ cells are primary sources for primitive hematopoiesis. It seems that precursors of primitive and definitive erythropoiesis arise simultaneously but that the definitive precursors require a period of maturation before they differentiated into blood cells. We suggest that a subset of endothelial cells is involved in primitive as well as definitive hematopoiesis during primate embryogenesis, and that α4-integrin marks the hemogenic subpopulation in primates.

Endoglin Is Required for Hemangioblast Development.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 138-138 ◽  
Author(s):  
Rita R. Perlingeiro
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Colony Formation ◽  
Time Course ◽  
Es Cells ◽  
Critical Role ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Embryoid Bodies ◽  
Wild Type

Abstract A critical role for endoglin (CD105) in early development has been demonstrated in mice deficient for this gene. Embryos homozygous for the endoglin mutation (eng−/−) fail to progress beyond 10.5 days postcoitum due primarily to vascular and cardiac abnormalities (Bordeau et al, 1999). Analysis of 9.5 dpc eng−/− embryos revealed abnormal vasculature and anemia of the yolk sac, suggesting that endoglin may be required for both blood and endothelial lineages. The hemangioblast, the bipotent precursor for hematopoietic and endothelial cells, can be assessed through the blast colony assay (BL-CFC) using a model system based on the in vitro differentiation of embryonic stem (ES) cells into embryoid bodies (EBs). To evaluate a role for endoglin in this early precursor, we differentiated eng−/−, eng+/−, and eng+/+ (wild-type) ES cells into EBs. At day 3 of EB differentiation, cells were disrupted and plated for blast colony formation in methylcellulose media containing vascular endothelial growth factor (VEGF), stem cell factor (SCF), and thrombopoietin (TPO). We found no difference in blast colony formation between heterozygous and wild-type ES cells. However, a significant reduction in the number of BL-CFCs was observed in eng−/− cells when compared to eng+/− or eng+/+ BL-CFCs (p < 0.001). Single eng−/−, eng+/−, and eng+/+ BL-CFCs gave rise to secondary hematopoietic colonies as well as endothelial cells, confirming their nature as hemangioblasts. These results suggest that although endoglin is required for hemangioblast development, its absence does not affect the bipotentiality of formed BL-CFCs. Since anemia was a feature of 9.5 dpc eng−/− yolk sac embryos, we also examined early erythropoiesis using the ES/EB system. For this purpose, eng−/−, eng+/−, and eng+/+ ES cells were differentiated into EBs for 4 days, at which time cells were disrupted and plated for primitive erythroid colonies (EryP) in methylcellulose media containing IL-3, IL-6, SCF, and Epo. We observed a reduction in the number of EryP colonies in eng−/− (p < 0.01) and eng+/− (p < 0.05) EBs when compared to controls (eng+/+). These results corroborate the anemia observed in vivo in the eng−/− embryos. We used RT-PCR and flow cytometry analysis to detect endoglin expression during a time course of EB differentiation. Endoglin is expressed in ES cells and disappears with differentiation. Expression re-appears at day 3 of differentiation, concomitantly with specification of the hemangioblast. Expression thereafter increases, correlating with mature endothelial cells at later time points. We did not find major differences in gene expression for Brachyury, Flk-1, Tie-2, embryonic and adult globins in a time course of EB differentiation for eng−/−, eng+/−, and eng+/+ ES cells. These data point out a role for endoglin, an ancillary receptor for several members of the transforming growth factor (TGF)-beta superfamily, in hemangioblast development.

CD41+ and Vascular Endothelial (VE)-Cadherin+ Cells: Two Developmental Origins of Hematopoietic Cell Lineages That Show Different Hematogenic Potentials.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3612-3612
Author(s):  
Kazuaki Hashimoto ◽  
Xin Huang ◽  
Yuri Shimoda ◽  
Guoyou Dai ◽  
Tetsuhiro Fujimoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Population ◽  
Stromal Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Yolk Sac ◽  
Mouse Embryos ◽  
Developmental Origins ◽  
Cell Lineages

Abstract It has been proposed that the definitive hematopoietic cell lineages are derived from hemogenic endothelial cells. Recently, CD41 was identified as an earliest cell surface marker of hematopoietic progenitor cells during mouse embryogenesis. We examined relationship between VE-cadherin+ hemogenic endothelial cells and CD41+ progenitors as developmental origins of hematopoietic cells by using in vitro differentiation system of ES cells as well as mouse embryos. FACS analyses on ES cells differentiating on OP9 stromal cells identified two cell populations, CD41+CD45− and CD41lowVE-cadherin+CD45−. The CD41+ cell population was derived from Flk1+ cells that represent lateral plate mesoderm but not from PDGFRa+ cells that represent paraxial mesoderm. CD41 expression on CD41+CD45− cells was weak at Day4 of ES cell culture. CD41+CD45− cells rapidly increased in number and CD41 expression became higher after Day5. CD45+ cells became detectable as a subpopulation of CD41+ cells two days after the appearance of the CD41+CD45− cell population. A significant proportion of the purified CD41lowVE-cadherin+CD45− cells differentiated to cells with CD41+CD45− phenotype in short-term culture, while CD41+CD45− cells did not differentiate into VE-cadherin+ cells. Unsurprisingly only CD41lowVE-cadherin+CD45− population had potential to produce endothelial cell colonies on OP9 cell layer. Liquid cultures and methylcellulose colony assay with a proper combination of cytokines showed that primitive erythroid colony forming cells were highly enriched in the CD41+CD45− cell population. CD41+CD45− cells also differentiated to Ter119+ definitive erythrocytes and Gr-1+ and Mac-1+ myeloid cells. In contrast, CD41lowVE-cadherin+CD45− cells produced only few hematopoietic cells in the same condition. However, CD41lowVE-cadherin+CD45− cells were capable of differentiating into multi-lineage hematopoietic cells including B lymphocytes when cultured with OP9 stromal cells. CD41+CD45− cells did not show any B lymphogenic potentials even when cultured with OP9 cells. We examined hemogenic potentials of phenotypically equivalent cells purified from mouse embryos. FACS analyses on cells dissociated from yolk sac and lower trunk of embryos proper revealed two distinct populations, CD41+CD45− cells and CD41−/lowVE-cadherin+CD45− cells. Both populations were already detectable in 8.5 dpc embryos. CD41−/lowVE-cadherin+CD45− cells but not CD41+CD45− cells produced endothelial cell colonies in vitro. The CD41−/lowVE-cadherin+CD45− cell population isolated from yolk sac was able to differentiate into multi-lineage hematopoietic cells when cultured with OP9 cells. However, the same population that was isolated from embryos proper had very poor potential to generate erythroid and myeloid cells although it still initiated robust production of B lymphocytes. Nevertheless, hemogenic activities of this population declined to undetectable level on 11.5 dpc. In contrast, CD41+CD45− cells isolated from yolk sac and embryos proper gave rise to multilineage hematopoietic cells and those potentials were stronger than that of yolk sac-derived CD41−/lowVE-cadherin+CD45− cells. Our results suggest that two distinct precursors, hemogenic endothelial cells and CD41+ progenitor cells, may contribute to the initiation of definitive hematopoiesis in mouse ontogeny, although activity of hemogenic endothelial cells in embryo proper might be unexpectedly limited.

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