Early lymph vessel development from embryonic stem cells.
Kreuger J, Nilsson I, Kerjaschki D, Petrova T, Alitalo K,
Department of Genetics and Pathology, Uppsala University, Sweden.
The purpose of this study was to establish a model system for lymph vessel development based on directed differentiation of
murine embryonic stem cells.
METHODS AND RESULTS:
Stem cells were aggregated to form embryoid bodies, and subsequently cultured in 3-dimensional collagen matrix for up to 18 days. Treatment with vascular endothelial growth factor (VEGF)-C and VEGF-A individually enhanced formation of lymphatic vessel structures, although combined treatment with VEGF-C and VEGF-A was most potent and gave rise to a network of LYVE-1, podoplanin, Prox1, and VEGF receptor-3 positive lymphatic vessel structures running parallel to and apparently emanating from, capillaries. In contrast, fibroblast growth factor-2, hepatocyte growth factor, or hypoxia had little or no effect on the development of the early lymphatics.
Further, cells of hematopoietic origin were shown to express lymphatic markers. In summary, different subpopulations of lymphatic endothelial cells were identified on the basis of differential expression of several lymphatic and blood vessel markers, indicating vascular heterogeneity.
We conclude that the present model closely mimics the early steps of lymph vessel development in mouse embryos.
PMID: 16543496 [PubMed - indexed for MEDLINE]
Differentiation of Lymphatic Endothelial Cells From Embryonic Stem Cells on OP9 Stromal Cells.
Kono T, Kubo H, Shimazu C, Ueda Y, Takahashi M, Yanagi K, Fujita N,
Tsuruo T, Wada H, Yamashita JK.
Molecular and Cancer Research Unit, HMRO and Department of Thoracic
Surgery, Graduate School of Medicine, Kyoto University, Japan;
Laboratory of Stem Cell Differentiation, Stem Cell Research Center,
Institute for Frontier Medical Sciences, Kyoto University, Japan;
Institute of Molecular and Cellular Biosciences, The University of
Tokyo, Japan; and PRESTO, Japan Science and Technology Agency, Japan.
The discovery of vascular endothelial growth factor C
(VEGF-C) and VEGF receptor-3 (VEGFR-3) has started to provide an
understanding of the molecular mechanisms of lymphangiogenesis. The
homeobox gene prox1 has been proven to specify lymphatic endothelial
cells (ECs) from blood ECs. We investigated the process of lymphatic EC
(LEC) differentiation using embryonic stem (ES) cells.
METHODS AND RESULTS:
VEGFR-2(+) cells derived from ES cells
differentiated into LECs at day 3 on OP9 stromal cells defined by the
expression of prox1, VEGFR-3, and another lymphatic marker podoplanin.
VEGFR-2(+) cells gave rise to LYVE-1(+) embryonic ECs, which were
negative for prox1 on day 1 but turned to prox1(+) LECs by day 3.
VEGFR-3-Fc or Tie2-Fc, sequestering VEGF-C or angiopoietin1 (Ang1),
suppressed colony formation of LECs on OP9 cells. However, addition of
VEGF-C and Ang1 in combination with VEGF to the culture of VEGFR-2(+)
cells on collagen-coated dishes failed to induce LECs. LEC-inducing
activity of OP9 cells was fully reproduced on paraformaldehyde-fixed
OP9 cells with the conditioned medium.
We succeeded in differentiating LECs from ES cells and revealed the requirements of VEGF-C, Ang1, and other unknown factors for LEC differentiation.
PMID: 16690875 [PubMed - as supplied by publisher]
Induction of lymphatic endothelial cell differentiation in embryoid bodies.
Liersch R, Nay F, Lu L, Detmar M.
Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
The molecular mechanisms that regulate the formation of the lymphatic vascular system remain poorly characterized. Whereas studies in embryonic stem (ES) cells have provided major new insights into the mechanisms of blood vessel formation, the development of lymphatic endothelium has not been previously observed. We established embryoid bodies (EBs) from murine ES cells in the presence or absence of lymphangiogenic growth factors. We found that lymphatic endothelial cells develop at day 18 after EB formation. These cells express CD31 and the lymphatic lineage markers Prox-1 and Lyve-1, but not the vascular marker MECA-32, and they frequently sprout from preexisting blood vessels. Lymphatic vessel formation was potently promoted by VEGF-A and VEGF-C but not by bFGF. Our results reveal, for the first time, that ES cells can differentiate into lymphatic endothelial cells, and they identify the EB assay as a powerful new tool to dissect the molecular mechanisms that control lymphatic vessel formation.
PMID: 16195336 [PubMed - indexed for MEDLINE]
http://www.ncbi.nlm.nih.gov/entrez/quer ... med_docsum
A chemically defined culture of VEGFR2+ cells derived from embryonic stem cells reveals the role of VEGFR1 in tuning the threshold for VEGF in developing endothelial cells.
Hirashima M, Ogawa M, Nishikawa S, Matsumura K, Kawasaki K, Shibuya M, Nishikawa S.
Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Japan.
Vascular endothelial growth factor (VEGF) is a major growth factor for developing endothelial cells (ECs). Embryonic lethality due to haploinsufficiency of VEGF in the mouse highlighted the strict dose dependency of VEGF on embryonic vascular development. Here we investigated the dose-dependent effects of VEGF on the differentiation of ES cell-derived fetal liver kinase 1 (Flk-1)/VEGF receptor 2(+) (VEGFR2(+)) mesodermal cells into ECs on type IV collagen under a chemically defined serum-free condition. These cells could grow even in the absence of VEGF, but differentiated mostly into mural cells positive for alpha-smooth muscle actin.
VEGF supported in a dose-dependent manner the differentiation into ECs defined by the expression of VE-cadherin, platelet-endothelial cell adhesion molecule 1 (PECAM-1)/ CD31, CD34, and TIE2/TEK. VEGF requirement was greater at late than at early phase of culture during EC development, whereas response of VEGFR2(+) cells to VEGF-E, which is a virus-derived ligand for VEGFR2 but not for Flt-1/VEGFR1, was not dose sensitive even at late phase of culture. Delayed expression of VEGFR1 correlated with increased dose dependency of VEGF.
These results suggested that greater requirement of VEGF in the maintenance than induction of ECs was due to the activity of VEGFR1 sequestering VEGF from VEGFR2 signal. The chemically defined serum-free culture system described here provides a new tool for assessing different factors for the proliferation and differentiation of VEGFR2(+) mesodermal cells.
PMID: 12406893 [PubMed - indexed for MEDLINE]