Evaluation of the use of an induced puripotent stem cell sheet for the construction of tissue-engineered vascular grafts - PubMed (original) (raw)

Evaluation of the use of an induced puripotent stem cell sheet for the construction of tissue-engineered vascular grafts

Narutoshi Hibino et al. J Thorac Cardiovasc Surg. 2012 Mar.

Abstract

Objective: The development of a living, tissue-engineered vascular graft (TEVG) holds great promise for advancing the field of cardiovascular surgery. However, the ultimate source and time needed to procure these cells remain problematic. Induced puripotent stem (iPS) cells have recently been developed and have the potential for creating a pluripotent cell line from a patient's own somatic cells. In the present study, we evaluated the use of a sheet created from iPS cell-derived vascular cells as a potential source for the construction of TEVG.

Methods: Male mouse iPS cells were differentiated into embryoid bodies using the hanging-drop method. Cell differentiation was confirmed by a decrease in the proportion of SSEA-1-positive cells over time using fluorescence-activated cell sorting. The expression of endothelial cell and smooth muscle cell markers was detected using real-time polymerase chain reaction (PCR). The differentiated iPS cell sheet was made using temperature-responsive dishes and then seeded onto a biodegradable scaffold composed of polyglycolic acid-poly-l-lactide and poly(l-lactide-co-ε-caprolactone) with a diameter of 0.8 mm. These scaffolds were implanted as interposition grafts in the inferior vena cava of female severe combined immunodeficiency/beige mice (n = 15). Graft function was serially monitored using ultrasonography. The grafts were analyzed at 1, 4, and 10 weeks with histologic examination and immunohistochemistry. The behavior of seeded differentiated iPS cells was tracked using Y-chromosome fluorescent in situ hybridization and SRY real-time PCR.

Results: All mice survived without thrombosis, aneurysm formation, graft rupture, or calcification. PCR evaluation of iPS cell sheets in vitro demonstrated increased expression of endothelial cell markers. Histologic evaluation of the grafts demonstrated endothelialization with von Willebrand factor and an inner layer with smooth muscle actin- and calponin-positive cells at 10 weeks. The number of seeded differentiated iPS cells was found to decrease over time using real-time PCR (42.2% at 1 week, 10.4% at 4 weeks, 9.8% at 10 weeks). A fraction of the iPS cells were found to be Y-chromosome fluorescent positive at 1 week. No iPS cells were found to co-localize with von Willebrand factor or smooth muscle actin-positive cells at 10 weeks.

Conclusions: Differentiated iPS cells offer an alternative cell source for constructing TEVG. Seeded iPS cells exerted a paracrine effect to induce neotissue formation in the acute phase and were reduced in number by apoptosis at later time points. Sheet seeding of our TEVG represents a viable mode of iPS cell delivery over time.

Copyright © 2012. Published by Mosby, Inc.

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Figures

Figure 1

Figure 1

Confirmation of differentiation of iPS cells in vitro. (a) The ratio of undifferentiated SSEA-1 positive cells by FACS analysis decreased over time after changing to differentiation medium. (b) Expression of endothelial markers (VEGF, PECAM and E-cadherin) and smooth muscle cell marker (calponin) in differentiated iPS cells.

Figure 2

Figure 2

Attached cell number and seeding efficiency of differentiated iPS cells seeded onto the scaffold. There was no significant difference for any incubation period.

Figure 3

Figure 3

Differentiated iPS cell sheet seeded TEVG. (a) Cell sheet creation from differentiated iPS cells. (b) The scaffold was wrapped with this cell sheet for seeding. (c) New seeding method using a differentiated iPS cell sheet significantly improved seeding efficiency compared with traditional pipette seeding. (d) Differentiated iPS cell sheet seeded TEVG was implanted in mouse IVC.

Figure 4

Figure 4

Histology of TEVG. (a) Seeded iPS cells were identified as Y chromosome FISH positive cells (red) at 1 week following implantation (Blue=DAPI). (b) At 4 weeks very small numbers of seeded cells were found. (c, d) At 10weeks, there was endothelialization with VWF (green) (c) and an inner layer with SMA (green) (d) positive cells at 10 weeks; however, no Y chromosome FISH positive seeded iPS cells (red) were seen. H&E staining (e) and ultrasonography (f) showed patent graft at 10 weeks.

Figure 5

Figure 5

Quantification of seeded differentiated iPS cells by quantitative real time PCR using probe for SRY. The number of seeded iPS cells decreased dramatically over time.

Figure 6

Figure 6

There was mixture of surviving (yellow arrows) and apoptotic (white arrows) seeded iPS cells at 1 week after graft implantation. (Green= TUNEL positive, Red= SRY FISH positive, Blue= DAPI

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