Erythropoiesis occurs through several waves during embryonic development.Although the source of the primitive wave is well characterized, the origin of erythrocytes later in embryogenesis is less clear due to overlaps between the different erythroid waves. Using the miR144/451-GFP mouse model to track cells expressing the erythroid microRNAs miR144/451, we identified cells co-expressing VE-Cadherin and GFP in the yolk sac between E9.5 and E12. This suggested the existence of hemogenic endothelial cells committed to erythropoiesis (Ery-HEC). We showed that these cells were capable of generating erythrocytes ex vivo and we demonstrated that the formation of Ery-HEC was independent of the Runx1 gene expression. Using transcriptome analysis, we demonstrated that these cells coexpressed endothelial and erythroid genes such as Hbb-bh1 and Gata1 but we were surprised to detect the primitive erythroid genes Aqp3 and Aqp8 suggesting the formation of primitive erythrocytes at a much later time point than initially thought.Finally, we showed that enforced expression of Gata1 in endothelial cells was enough to initiate the erythroid transcriptional program.
Erythropoiesis occurs through several waves during embryonic development.Although the source of the primitive wave is well characterized, the origin of erythrocytes later in embryogenesis is less clear due to overlaps between the different erythroid waves. Using the miR144/451-GFP mouse model to track cells expressing the erythroid microRNAs miR144/451, we identified cells co-expressing VE-Cadherin and GFP in the yolk sac between E9.5 and E12. This suggested the existence of hemogenic endothelial cells committed to erythropoiesis (Ery-HEC). We showed that these cells were capable of generating erythrocytes ex vivo and we demonstrated that the formation of Ery-HEC was independent of the Runx1 gene expression. Using transcriptome analysis, we demonstrated that these cells coexpressed endothelial and erythroid genes such as Hbb-bh1 and Gata1 but we were surprised to detect the primitive erythroid genes Aqp3 and Aqp8 suggesting the formation of primitive erythrocytes at a much later time point than initially thought.Finally, we showed that enforced expression of Gata1 in endothelial cells was enough to initiate the erythroid transcriptional program..
Stem/progenitor cells hold a great promise for application in several therapies due to their unique biological characteristics. With the purpose of harnessing these cells full potential in cell-or gene-based therapies it might be advantageous to enhance some of their features through gene delivery strategies. Accordingly, we are interested in developing efficient and safe methodologies to genetically engineer stem cells, boosting their therapeutic efficacy in Regenerative Medicine. In our work, delivery of plasmid DNA to human Bone Marrow Mesenchymal Stem Cells (BM-MSC) was optimized by lipofection and by a recently available microporation technique and no effect was observed in their immunophenotypic characteristics or differentiative potential. After lipofection similar number of plasmid copies was determined at different cell passages. Importantly, cell proliferation kinetics slowed down due to the presence of plasmid. Overall, we believe our findings are extremely useful towards the maximization of gene delivery to human MSC, without compromising cell function and viability.
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