In this study we demonstrated the feasibility of using a differentiated iPS cell sheet as an alternative cell source for TEVG creation. Most of seeded iPS cells disappeared as a result of apoptosis at later time points, suggesting that seeded iPS cells exerted a paracrine effect to induce neotissue formation in the acute phase.
Cells are one of the key factors involved in vascular regeneration and various cell types have been used for vascular tissue engineering. Stem cells or progenitor cells can differentiate into vascular cells that form new vessels or tissues. Mesenchymal stem cells (17
) and endothelial progenitor cells (18
) reside in the bone marrow and have been studied most for their vascular regeneration potential. Bone marrow derived stem cells have undergone the most translational and human studies of all stem cell approaches. Clinical trials performed to date show little increased risk associated with their therapeutic use in humans (19
). On the other hand, there are limitations of bone marrow derived stem cell therapy. The typical surface markers used to isolate these stem cells result in a mixed population of cells that have not been completely characterized (21
). In addition, in the patients who most need stem cell therapy, these cells are rare, have limited replicative capacity, and are often dysfunctional, especially in patients who have the conditions typically associated with vascular disease, such as older age and diabetes mellitus, which impair angiogenic functionality (21
Our previous studies have demonstrated the feasibility of creating TEVGs by seeding bone marrow mononuclear cells onto biodegradable tubular scaffolds (2
). These cells disappeared rapidly after implantation and exert a paracrine effect to facilitate neotissue creation (26
). In addition, we have shown that it takes 10 weeks for complete neovessel formation with endothelialization and smooth muscle layer creation. Compared with bone marrow derived stem cells, ESCs have the advantage of pluripotentiality and greater proliferative capacity, suggesting they might be a useful source for cell seeding, but the clinical use of ESCs remains conflicted by ethical debate and immunologic barriers. As an alternative to ESCs, iPS cells are generated by inducing forced expression of certain stem cell-associated genes in non-pluripotent cells (11
). iPS cells are similar to ESCs in many respects and provide the potential to generate donor-specific pluripotent cells. Interestingly, recent reports have shown that murine iPS cells can be differentiated into cardiovascular cells needed to repair heart and blood vessels and may represent a valuable cell source for vascular regeneration (29
). Therefore, we investigated the application of stem cell technology with the idea of creating a patient’s own stem cells in order to promote earlier and better vascular neotissue formation.
Seeding methods for the delivery of iPS cells are diverse, and no method has been clearly shown to be superior in either promoting seeding efficiency or improving long-term graft function (31
). To date, the most common method used in the construction of TEVGs is static seeding, in which a concentrated cell suspension is passively introduced onto a scaffold. This technique has several limitations that result in low efficiency seeding and minimal cell penetration of scaffold walls. As we demonstrated in this study, average seeding efficiency is around 10%. Okano and co-workers developed a novel cell sheet system to engineer a graft that contracts in three dimensions (32
). The trick to obtain a stable cell sheet was the use of temperature-responsive culture surfaces. A stable cell sheet of single-cell thickness can be harvested without losing the intercellular connections and then can be transferred to a second and third cell sheet. In order to increase the delivery of iPS cells with our TEVG, we applied this cell sheet technology for differentiated iPS cells and seeding efficiency was found to be significantly improved.
Although the sheet seeding method led to a dramatic improvement in seeding efficiency, our results in vivo suggested that seeded iPS cells disappeared in the acute phase by apoptosis. While the cell number remaining on the graft after implantation was higher than our previous study (26
), no engraftment of seeded cells was seen in our neovessels at 10 weeks. Some groups, however, have reported that seeded iPS cells can engraft with tissue in other models. In a study of rat arterial TEVGs fabricated with biodegradable tubular scaffolds and seeded with muscle-derived stem cells (MDSCs), the engraftment of seeded MDSCs in the vascular tissue was identified by the direct comparison between histology and LacZ positive seeded cell images (35
). In a murine model of ischemic myocardium, transplanted endothelial cells differentiated from ESCs were tracked using bioluminescence imaging, which showed persistence of the cells up to 8 weeks later. Echocardiography in this study revealed improved systolic function in hearts injected with ESC-ECs compared with vehicle (36
). On the other hand, other groups have reported findings consistent with the results of our study. In a study of transplantation of human ESC-derived cardiomyocytes into ischemic murine myocardium, implanted cells died soon after transplantation into the infarcted heart. This problem was also found in other cell therapies for diabetes (37
), Parkinson’s disease (39
) and muscular dystrophy (41
). Since the death of these transplanted cells was multifactorial in origin, varied and complex interventions were required for the engraftment of cells in tissue. A cocktail of pro-survival factors including matrigel and inhibitors of apoptosis was proposed for targeting key components of potential cell death pathways (43
). Although the fate of seeded cells is controversial, the further elucidation of pathways that cause iPS cell death would be an important next step to better understand and enhance the role of seeded iPS cells in the creation of improved TEVGs.
The heterogeneity inherent to the epigenetics and gene expression profiles of pluripotent cells raises concern for pleiotropic outcomes such as teratoma formation (44
). Our study showed that teratomas formed in 25% of the grafts. In order to prevent the formation of teratomas, further purification of differentiated iPS cells will be required. Isolation using immunomagnetic beads or culture with growth factors such as VEGF and PDGFBB are possible methods for improvement in purification(45
). However, no method to properly direct this differentiation has yet been mastered. Studies have demonstrated that no growth factor causes differentiation along a single cell line and, in contrast, differentiation has been shown to be spontaneous(47
In conclusion, differentiated iPS cells provide an alternative and attractive cell source for constructing TEVGs using the sheet engineering technique. Taking into consider that the number of seeded iPS cell decreased over time as a result of apoptosis in the early phase, seeded iPS cells could work as a paracrine effect to induce neotissue formation in the acute phase. However, further study such as cytokine array or microarray is required to show the exact mechanism of paracrine effect. There remains some possibility of teratoma formation in the present differentiation method, which represents a drawback for clinical applications of this approach. Further study of the inhibition of apoptosis of seeded cells and the improvement in iPS cell purification procedures will enable the design and creation of the next generation of TEVGs.