Efficient Ex Vivo Expansion of Highly Purified Human Umbilical Cord Blood-Derived Very Small CD34⁺lin^-CD45^- Stem Cells into Functional Endothelial Cells in Vitro in Chemically Identified, Feeder Layer-Free Medium Supplemented with Nicotinamide

Background. There is an ongoing search for multipotent stem cells from umbilical cord blood (UCB) with trans-germ layer differentiation potential that can be employed in repairing damaged organs and also expanded into transplantable hematopoietic stem cells (HSCs) and endothelial progenitor cells (EPCs). The existence of such cells in postnatal life could also revive the concept of hemangioblasts or hemangioblast-like cells in adult hematopoietic organs. Our group was the first to isolate a population of small CD34⁺CD133⁺lin^-CD45^- early-development stem cells from human hematopoietic tissues, including UCB. Based on the validated expression of early-development markers, these cells were named "very small embryonic-like stem cells" (VSELs, Circulation Res 2019; 124:208-210). Currently, more than 25 independent groups worldwide who have carefully followed the multicolor-staining cell-sorting strategy described by us (Current Protocols in Cytometry 2010, 9.29.1-9.29.15) have successfully isolated these cells and demonstrated their in vivo contribution to all three germ layer lineages. Thus, VSELs could be very useful in regenerative medicine in the field of angiogenesis, and UCB is an attractive source, with easy accessibility and tolerance to allogenic grafts. However, the low number of these cells in UCB and their quiescence are limiting factors. Therefore, in vitro differentiation of VSELs into endothelial progenitor cells (EPCs) would allow improvement in the ability to expand endothelial cells and could represent a clinically relevant alternative to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPS) for cell therapy without ethical problems and undesirable side effects. Hypothesis. We hypothesized that UCB-purified, very small, early-developmentCD34⁺lin^-CD45^-stem cells can be ex vivo expanded into functional EPCs. Materials and Methods. VSELs highly purified by FACS were expanded into EPCs in pro-angiogenic medium supplemented with mesodermic differentiation factors and then endothelial differentiation factors in the presence of nicotinamide and UM171. In parallel, we expanded EPCs from MNCs isolated from the same UCB units by employing a classical protocol (Methods in Enzymology 2008, 445:303-29). The EPC nature of the expanded VSEL-derived cells was confirmed by the expression of typical EPC markers as well as by in vitro angiogenic assays. Results. Our differentiation cocktail allowed us to differentiate and expand VSELs into EPCs. In our expansion medium (Figure 1), the very small, round VSELs smaller than 6 mm in diameter proliferated and differentaited over time into larger and extended cells with a cobblestone morphology similar to the EPC control cells, and we confirmed their endothelial characteristics by cytometry analysis. Like EPCs, VSEL-derived EPCs were positive for CD31, CD144, KDR, and CD105 and negative for mesenchymal surface markers, such as CD90. They also performed similarly to EPCs in classical vasculogenic tests, including adhesion, proliferation, migration, and tubulogenesis assays. Conclusions. This work shows, for the first time, efficient VSEL differentiation into functional endothelial cells with vasculogenic properties without the help of co-culture over feeder-layers or viral vectors in medium supplemented with nicotinamide and UM171. These findings allow us to propose these cells as an interesting cell therapy product. These results also reopen the question of the existence of hemangioblast-like cells in postnatal tissues. We are currently testing these cells in vivo in model of hind limb ischemia.

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