VEGF Induces Differentiation of Functional Endothelium From Human Embryonic Stem Cells. Implications for Tissue Engineering

Marilyn B. Nourse, Daniel E. Halpin, Marta Scatena, Derek J. Mortisen, Nathaniel L.

Tulloch, Kip D. Hauch, Beverly Torok-Storb, Buddy D. Ratner, Lil Pabon and Charles E. Murry

Introduction:

Human embryonic stem cells (hESCs) have shown promise in the application for regenerating tissues with specific cell types. Currently however, the obstacles that arise relate to the inefficiency of the current techniques used to promote and distinguish the functional endothelial cells from differentiating hESCs. This article attempts to use a vascular endothelial grown factor (VEGF) as treatment and demonstrated a 4-5 fold enrichment of endothelial cells which in turn were implanted to form vessels and deemed viable in vivo. This implication will facilitate future endeavors involving tissue-engineered implants.

Methods:

Undifferentiated hESCs were allowed to turned into embryoid bodies (EBs) by separated confluent cultures into small clumps. VEGF-induced and control EBs, human umbilical vein endothelial cells (HUVECs), were then dispersed into single cells and stained with fluorescent conjugated antibodies (CD31, VECad, vWF, CD45[negative control]). The EBs were grown for 4 or 14 days and then monolayers of cells (HUVECs and DC31) were serum starved and then stimulated with TNFa. The RNA collected was quantified via RT-PCR and the protein analysis was taken via Western blotting. The cells were also assayed for tubule formation and colony forming ability (methylcellulose assay). Three million CD31 cells were mixed into a gel/culture medium under standard tissue culture conditions. The porous scaffolds were then prepared with hESC-influenced endothelial cells seeded. The scaffolds were later implanted into rats and processed for histology after ten days. The fluorescent images were lastly attained by confocal microscopy.

Results/Discussion:

Figure 1 portrays the induction of endothelium with VEGF. The test of cell differentiation showed that the treatment with VEGF resulted in more protein than standard cultures.

An increase of the dosage of VEGF also correlated to a positive expression until around 50ng/mL.

Figure 2 illustrates the immunofluorescence of the VEFG-induced EBs with the various markers. The plots for day 10 and 14 was to justify that while all the other markers coexpressed, CD45 never was detected in all conditions and time variables. CD45 is a marker for hematopoetic stem cells which indicate unwanted differentiation.

Figure 3 shows that although VEGF induces the differentiation in favor of endothelium it does not effect the proliferation in EBs.

Figure 4 further confirmed the cells' morphology and also that they were negative for CD45 and positive for the markers CD31, VeCAD, and vWF which is what was anticipated.

VEGF was shown to have a 4.7-fold increase in the number of differentiating endothelial cells but as for proliferation analysis, it resulted in no difference.

Figure 5 show that in vivo hESC-derived endothelial develop into robust vascular networks. These are different magnifications of CD31 stained A,C,D and unstained B in different magnifications.

Discussion/Applications:

Overall, this article discovered a novel and important finding of how to induce differentiation for functional endothelium. This could potentially help with the process of differentiation of mesodermal precursor cells that could expand the cell populations in hESC cultures and create a useful source for cardiac tissue engineering applications. Also ECs derived from hESC may improve neovascularization in transplant and scaffolds for tissue engineering. if combined with collagen gels in vivo, they could help organize into vascular networks as well.

The only critique for this article is that they do not indicate whether or not the endothelial cells achieve sustained expansion and stability of vascular cells. Vascular commitment is essential in making sure that the grafts, transplants, or other engineering products are sustainable in potential patients.