Derivation of endothelial cells and formation of microvasculature from mouse embryonic stem cells
January 15-19, 2017
Mouse Embryonic Stem Cells (mESC) are pluripotent cells derived from inner cells mass of blastocyst. mES cells have the ability of in vitro self-renew, preserving the potential to generate the three embryonic layers, mainly due to the expression of important regulators of pluripotency such as Oct 3/4 and Nanog. mES cells have been widely used in different experimental models of regenerative medicine and tissue engineering. The objective of this study was to derive a monolayer protocol to induce the differentiation of mESC into vascular precursor cells (VPC) and endothelial specialized cells (ESC). The mESC were maintained according to the protocol reported by Mondragón et al., (2011) and the differentiation method was modified of the protocols reported by McCloskey et al., (2006) and Hatano et al., (2013). We compared the differentiation and expansion of the VPC derived from mESC in Collagen Type I and type IV (used as extracellular matrix), as well as two VEGF concentrations (10 ng∙ml-1 and 30 ng∙ml-1). The collagen Type IV probes the optimal matrix, moreover no differences were found in the use of both VEGF concentrations. We assessed the expression of different proteins as markers for: pluripotent cells (Oct ¾+), vascular precursor cells (Flk-1+ and PECAM+) and endothelial specialized cells (Ve-Cadherin+) by immunocytochemistry at different time points (120h, 360h, 600h and 840h) of differentiation. Population dynamics during differentiation kinetics, showed that there is an initial expansion of pluripotent cells (Oct4+) from 0 to 120 h; meanwhile the production and expansion of vascular precursor cells (Flk-1+ and PECAM+) was observed from 120 h to 360 h (Ve-Cadherin+); finally the “maturation” process and expansion of specialized endothelial cells was observed from 360 h to 840 h. At the end of the differentiation process (840 h), we detected pluripotent cells (Oct ¾+: 3.1% ± 1.1%), vascular progenitor cells (Flk-1+: 20.2% ± 6.1%; & PECAM+: 16.4% ± 4.0%) and endothelial specialized cells (VE- Cadherin+: 23.85% ± 4.5%). The overall behavior of population dynamics throughout the differentiation process showed three stages: The expansion of pluripotent cells, the generation and expansion of endothelial progenitors and the maturation of endothelial cells. Specific growth rate (SGR) was calculated for each of the stages of the differentiation; importantly the specific growth rate of pluripotent cells (SGR= 0.0226 h-1) was 1 log higher than the values of for the expansion of both endothelial progenitors (SGR= 0.0094 h-1) and endothelial cells (SGR= 0.0011 h-1). Microvasculature formation was observed at 840 h of differentiation process; transmission electron microscopy analyses allowed us to identify a large number of desmosome connections (adherent junctions) between endothelial cells, and the formation of communicative cell junctions, as well as the formation of small and wider intercellular channels at intercellular junction.
Hatano,R., Mercurio, K., Luna, J., Glaser, D., Leppert, V. & McCloskey, K. (2013). Endothelial cells derived from embryonic stem cells respond to cues from topographical surface patterns. Journal of Biological Engineering. 7:18. 1-12.
McCloskey, K., Smith, D., Jo, H. & Nerem, R. (2006) Embryonic Stem Cell Derived Endothelial Cells May Lack Complete Functional Maturation in vitro. Journal of vascular research. 43(5): 411- 421.
Mondragón-Terán, P., Baboo, J., Mason, C., Lye, G. & Veraitch, F. (2011). The Full Spectrum of Physiological Oxygen Tensions and Step-Changes in Oxygen Tension Affects the Neural Differentiation of Mouse Embryonic Stem Cells. Biotechnology Progress. 27(6): 1700-1708.
Alan Jesús Gómez-Calderon, Juan Antonio Suárez-Cuenca, and Paul Mondragón-Teran, "Derivation of endothelial cells and formation of microvasculature from mouse embryonic stem cells" in "Scale-up and Manufacturing of Cell-based Therapies V", Tom Brieva, Celgene Cellular Therapeutics William Miller, Northwestern University Chris Mason, University College London Eds, ECI Symposium Series, (2017). http://dc.engconfintl.org/cellbasedtherapies_v/11