VEGFR-3 and CD133 identify a population of CD34+ lymphatic/vascular endothelial precursor cells Short title for running head: VEGFR-3+ lymphatic/vascular endothelial precursors

Human CD133(AC133)+CD34+ stem and progenitor cells derived from fetal liver and bone marrow, and blood incorporate a functional population of circulating endothelial precursor cells. Vascular endothelial growth factor receptor 3 (VEGFR-3) regulates cardiovascular development, and physiological and pathological lymphangiogenesis and angiogenesis. However, the origin of VEGFR-3+ endothelial cells (ECs) and the mechanisms by which these cells contribute to postnatal physiological processes are not known, and the possible existence of VEGFR-3+ lymphatic or vascular EC progenitors has not been studied. Using monoclonal antibodies to the extracellular domain of VEGFR-3, we show that 11±1% of CD34+ cells isolated from human fetal liver, 1.9±0.8% CD34+ cells from human cord blood, and 0.2±0.1% of CD34+ cells from healthy adult blood donors are VEGFR-3+. CD34+VEGFR-3+ cells from fetal liver coexpress the stem/precursor cell marker CD133(AC133). Because mature endothelial cells do not express CD133, coexpression of VEGFR-3 and CD133 on CD34+ cells identifies a unique population of stem and progenitor cells. Incubation of isolated CD34+VEGFR-3+ cells in endothelial cell growth medium resulted in a strong proliferation (40-fold in 2 weeks) of nonadherent VEGFR-3+ cells. Plating of these cells resulted in the formation of adherent VEGFR-3+Ac-LDL+ (acetylated low density lipoprotein+) EC monolayers expressing various vascular and lymphatic endothelial-specific surface markers including CD34, VE-cadherin, CD51/61, CD105, LYVE-1, and podoplanin. These data demonstrate that human CD34+CD133+ cells expressing VEGFR-3 constitute a phenotypically and functionally distinct population of endothelial stem and precursor cells that may play a role in postnatal lymphangiogenesis and/or angiogenesis. The corresponding author's e-mail address: [email protected]; [email protected]