Characterization of long term repopulating endothelial progenitor cells in the mouse embryo

Resumen del trabajo presentado al Workshop Current Trends in Biomedicine: "The Hemato-Vascular System: Development and Disease" celebrado en Baeza, Jaén (España) del 21 al 23 de octubre de 2013.The development of the hematopoietic and vascular systems is interconnected. This is reflected by several aspects: they share a hemangioblast progenitor; specialized endothelial populations are capable of hematopoiesis; and also blood cell subsets are critical for vascular remodelling. Moreover, hematopoietic progenitors from the adult bone marrow can contribute to endothelial cells upon transplantation. We have reported that E12 foetal liver cells expressing the hemato/vascular Stem Cell Leukaemia (SCL) gene derived vector, the SCL-3'Enh-PLAP (SCL-PLAP), contained long term repopulating hematopoietic stem cells (LTR-HSCs) and endothelial progenitor cell activity (LTR-EPC). We described LTR-EPC activity as the capacity of progenitor cell population to stably repopulate/colonize vascular beds in targeted organs when transplanted into busulfan treated new-borns or adult irradiated mice. In the study we present here we wanted to determine when LTR-EPC potential emerged during development and characterize its phenotype and in vitro capacities to evaluate its ontogenic relation to blood and other endothelial populations. To this end, we performed transplantation of cells from different embryonic/foetal hematopoietic locations followed by long-term donor cell lineage tracing and quantification of vascular contribution on liver sections. LTR-EPC activity was mostly restricted to the foetal liver, starting at stage E11. Some activity was also identified in E12 AGM but was absent from the YS and placenta. Further FACS cell sorting according to SCL-PLAP and Ve-cadherin (Ve) expression, revealed E12 FL LTR-EPC activity restricted to the SCL+Ve+ population. Moreover, by adding the pan-leukocyte marker CD45 we showed that multi-organ LTR-EPC activity was ascribed to the SCL+Ve+CD45- population, whereas the HSCs containing SCL+Ve+CD45+ cells contributed sporadically to endothelial cells. Using a panel of endothelial, progenitor and hematopoietic associated markers showed lyve1 as a distinctive receptor expressed in the majority of E12 FL SCL+ Ve+CD45- cells. Interestingly, the Ve+CD45-Lyve1+ population increased with developmental time in the FL but constituted a minor population within the AGM Ve+CD45- cells, suggesting lyve1 as a marker for emergence of LTR-EPC activity in foetal liver. Further in vitro analysis using CFU-C assays and OP9 co-cultures for endothelial and hemogenic activity revealed that the Ve+CD45-Lyve1+ population presented a high endothelial tube formation capacity and lacked hematopoietic and hemogenic potential. In summary, our results indicate that the LTR-EPC activity is ascribed to the foetal liver SCL+Ve+CD45- population showing its distinctive endothelial identity and divergence from the hematopoietic lineage. These findings should provide new insights into the development and function of tissue-associated hemato-vascular progenitors with transplantation capabilities characterized by the expression of the SCL cis-regulatory sequences and organ associated endothelial markers.Peer Reviewe