Identification and Characterization of Hemoangiogenic Progenitors During Cynomolgus Monkey Embryonic Stem Cell Differentiation

We identified intermediate‐stage progenitor cells that have the potential to differentiate into hematopoietic and endothelial lineages from nonhuman primate embryonic stem (ES) cells. Sequential fluorescence‐activated cell sorting and immunostaining analyses showed that when ES cells were cultured in an OP9 coculture system, both lineages developed after the emergence of two hemoangiogenic progenitor‐bearing cell fractions, namely, vascular endothelial growth factor receptor (VEGFR)‐2high CD34− and VEGFR‐2high CD34+ cells. Exogenous vascular endothelial growth factor increased the proportion of VEGFR‐2high cells, particularly that of VEGFR‐2high CD34+ cells, in a dose‐dependent manner. Although either population of VEGFR‐2high cells could differentiate into primitive and definitive hematopoietic cells (HCs), as well as endothelial cells (ECs), the VEGFR‐2high CD34+ cells had greater hemoangiogenic potential. Both lineages developed from VEGFR‐2high CD34−or VEGFR‐2high CD34+ precursor at the single‐cell level, which strongly supports the existence of hemangioblasts in these cell fractions. Thus, this culture system allows differentiation into the HC and EC lineages to be defined by surface markers. These observations should facilitate further studies both on early developmental processes and on regeneration therapies in human.

[1]  J. D. Engel,et al.  GATA Motifs Regulate Early Hematopoietic Lineage-Specific Expression of the Gata2 Gene , 2005, Molecular and Cellular Biology.

[2]  A. Elefanty,et al.  SCL/Tal-1 is essential for hematopoietic commitment of the hemangioblast but not for its development. , 2005, Blood.

[3]  Lisheng Wang,et al.  Endothelial and hematopoietic cell fate of human embryonic stem cells originates from primitive endothelium with hemangioblastic properties. , 2004, Immunity.

[4]  M. Shibuya,et al.  Development of primitive and definitive hematopoiesis from nonhuman primate embryonic stem cells in vitro , 2004, Development.

[5]  A. Rouleau,et al.  VEGF-A165 augments erythropoietic development from human embryonic stem cells. , 2004, Blood.

[6]  Takumi Miura,et al.  Long‐term culture of human embryonic stem cells in feeder‐free conditions , 2004, Developmental dynamics : an official publication of the American Association of Anatomists.

[7]  N. Nakatsuji,et al.  Electroporation of cynomolgus monkey embryonic stem cells , 2003, Genesis.

[8]  L. Liao,et al.  Hemangioblastic characteristics of fetal bone marrow-derived Flk1(+)CD31(-)CD34(-) cells. , 2003, Experimental hematology.

[9]  S. Nishikawa,et al.  Different Differentiation Kinetics of Vascular Progenitor Cells in Primate and Mouse Embryonic Stem Cells , 2003, Circulation.

[10]  C. Peschle,et al.  Autocrine-paracrine VEGF loops potentiate the maturation of megakaryocytic precursors through Flt1 receptor. , 2003, Blood.

[11]  Patrick W. Faloon,et al.  Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse. , 2003, Genes & development.

[12]  P. Kingsley,et al.  Lineage analysis of the hemangioblast as defined by FLK1 and SCL expression , 2002, Development.

[13]  C. Peschle,et al.  Identification of the hemangioblast in postnatal life. , 2002 .

[14]  Sridevi Devaraj,et al.  Demonstration That C-Reactive Protein Decreases eNOS Expression and Bioactivity in Human Aortic Endothelial Cells , 2002, Circulation.

[15]  Shulamit Levenberg,et al.  Endothelial cells derived from human embryonic stem cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[16]  S. Nishikawa,et al.  Step‐wise divergence of primitive and definitive haematopoietic and endothelial cell lineages during embryonic stem cell differentiation , 2001, Genes to cells : devoted to molecular & cellular mechanisms.

[17]  T. Tada,et al.  Establishment of embryonic stem cell lines from cynomolgus monkey blastocysts produced by IVF or ICSI , 2001, Developmental dynamics : an official publication of the American Association of Anatomists.

[18]  James A. Thomson,et al.  Hematopoietic colony-forming cells derived from human embryonic stem cells , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[19]  J. Thomson,et al.  Bone morphogenetic protein 4 induces efficient hematopoietic differentiation of rhesus monkey embryonic stem cells in vitro. , 2001, Blood.

[20]  K. Shitara,et al.  and surface marker for the lineage of monocyte-macrophages in humans Flt-1 , vascular endothelial growth factor receptor 1 , is a novel cell , 2001 .

[21]  K. Ozawa,et al.  Gene Transfer into Nonhuman Primate Hematopoietic Stem Cells: Implications for Gene Therapy , 2001, Stem cells.

[22]  F. Tashiro,et al.  Upgrading of flow cytometric analysis for absolute counts, cytokines and other antigenic molecules of cynomolgus monkeys (Macaca fascicularis) by using anti-human cross-reactive antibodies. , 2000, Experimental animals.

[23]  G. Breier.  Endothelial receptor tyrosine kinases involved in blood vessel development and tumor angiogenesis. , 2000, Advances in experimental medicine and biology.

[24]  B. Alter,et al.  Embryonic hemoglobins are expressed in definitive cells. , 1999, Blood.

[25]  A. Thrasher,et al.  Detailed characterization of the human aorta‐gonad‐mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer , 1999 .

[26]  S. Nishikawa,et al.  Maturation of embryonic stem cells into endothelial cells in an in vitro model of vasculogenesis. , 1999, Blood.

[27]  A. Thrasher,et al.  Detailed characterization of the human aorta-gonad-mesonephros region reveals morphological polarity resembling a hematopoietic stromal layer. , 1999, Developmental dynamics : an official publication of the American Association of Anatomists.

[28]  M. Yamamoto,et al.  GATA-1 regulates growth and differentiation of definitive erythroid lineage cells during in vitro ES cell differentiation. , 1998, Blood.

[29]  C. Dulac,et al.  Molecular identity of hematopoietic precursor cells emerging in the human embryo. , 1998, Blood.

[30]  J. Jimeno,et al.  Antigenic profiles of endothelial and hemopoietic lineages in murine intraembryonic hemogenic sites. , 1998, Developmental and comparative immunology.

[31]  S. Nishikawa,et al.  Progressive lineage analysis by cell sorting and culture identifies FLK1+VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. , 1998, Development.

[32]  S. Orkin,et al.  Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development. , 1998, Genes & development.

[33]  M. Kennedy,et al.  A common precursor for hematopoietic and endothelial cells. , 1998, Development.

[34]  T. Enver,et al.  CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis. , 1997, Blood.

[35]  Janet Rossant,et al.  A Requirement for Flk1 in Primitive and Definitive Hematopoiesis and Vasculogenesis , 1997, Cell.

[36]  L. Zon,et al.  Genetics of erythropoiesis: induced mutations in mice and zebrafish. , 1997, Annual review of genetics.

[37]  D. Luton,et al.  Aorta-associated CD34+ hematopoietic cells in the early human embryo. , 1996, Blood.

[38]  C. Begley,et al.  Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[39]  Janet Rossant,et al.  Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice , 1995, Nature.

[40]  S. Orkin,et al.  Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL , 1995, Nature.

[41]  M. Wiles,et al.  Hematopoietic commitment during embryonic stem cell differentiation in culture. , 1993, Molecular and cellular biology.

[42]  G. Keller,et al.  Multiple hematopoietic lineages develop from embryonic stem (ES) cells in culture. , 1991, Development.

[43]  R Kemler,et al.  The in vitro development of blastocyst-derived embryonic stem cell lines: formation of visceral yolk sac, blood islands and myocardium. , 1985, Journal of embryology and experimental morphology.

[44]  D. Solter,et al.  Stage‐specific embryonic antigens (SSEA‐3 and ‐4) are epitopes of a unique globo‐series ganglioside isolated from human teratocarcinoma cells. , 1983, The EMBO journal.

[45]  P. Murray.  The development in vitro of the blood of the early chick embryo , 1932 .

[46]  F. Sabin.  Studies on the origin of blood vessels and of red corpuscles as seen in the living blastoderm of the chick during the second day of incubation , 1920 .