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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 this study we evaluated the use of a sheet created from iPS cell-derived vascular cells as a potential source for the construction of TEVGs.
Male mouse iPS cells were differentiated into embryoid bodies with the hanging-drop method. Cell differentiation was confirmed by a decrease in the proportion of SSEA-1 positive cells over time using FACS. Expression of endothelial cell and smooth muscle cell markers was detected by Real-Time PCR. The differentiated iPS cell sheet was made using temperature-responsive dishes and then seeded onto a biodegradable scaffold composed of PGA-P(CL/LA) with a diameter of 0.8mm. These scaffolds were implanted as interposition grafts in the inferior vena cava of female SCID/bg mice (N=15). Graft function was serially monitored using ultrasound. The grafts were analyzed at 1, 4, and 10 weeks with histology and immunohistochemistry. The behavior of seeded differentiated iPS cells was tracked using Y-chromosome FISH and SRY Real- Time PCR.
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. Histological evaluation of the grafts demonstrated endothelialization with VWF and an inner layer with SMA and calponin positive cells at 10 weeks. The number of seeded differentiated iPS cells was found to decrease over time by Real-Time PCR (42.2% at 1wk, 10.4% at 4wks, 9.8% at 10wks). A fraction of the iPS cells were found to be TUNEL positive at 1 week. No iPS cells were found to co-localize with VWF or SMA positive cells at 10 weeks.
Differentiated iPS cells offer an alternative cell source for constructing TEVGs. 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.
Surgeons and scientists have looked to tissue engineering as a means of creating blood vessel substitutes with the ability to repair, remodel, and grow(1). The development of a tissue engineered vascular graft (TEVG) with bone marrow-derived mononuclear cells, differentiated smooth muscle cells (SMC), or endothelial cells (EC) seeded onto a biodegradable tubular scaffold, has resulted in living vascular conduits with properties that mimic those of a native vessel (2–5). We translated this basic science research and performed the first clinical trial evaluating the use of TEVGs in congenital heart surgery(6). This pilot study demonstrated not only that was it feasible to successfully implant TEVGs in humans, but also that this technology was safe and efficacious(7, 8).
The ultimate source of cells for seeding the TEVG, however, remains problematic. In addition, little is known about the mechanisms of seeded cell engraftment that underlie the formation of vascular neotissue in vivo. In order to explore the cellular and molecular mechanisms essential for neovessel formation, we developed a miniaturized version of the tissue engineered scaffold used in our clinical study in order to enable TEVG implantation in a murine model(9). This model showed that seeded bone marrow mononuclear cells (BMMNC) exerted a paracrine effect to induce neotissue formation and disappeared in the acute phase soon after implantation. Although BMMNC were appropriate for TEVG creation in a low-pressure venous model, a stronger contribution of seeded cells seems to be required for appropriate neovessel formation in high-pressure systems.
Embryonic stem cells (ESC) have the potential to differentiate into various cell types and ESCs may provide a source of cells for seeding a variety of tissue engineering constructs(10). Since clinical use of ESC remains challenging due to ethical and immunologic problems, induced pluripotent stem (iPS) cells were developed by inducing forced expression of certain stem cell-associated genes in non-pluripotent cells(11).
In this study, we sought to determine if seeded iPS cells could differentiate into vascular neotissue and contribute to neovessel formation in a murine model.
Induced pluripotent stem cells were purchased from RIKEN BRC (Tokyo, Japan)(12) and maintained on mitomycin-treated embryonic feeders in DMEM medium supplemented with 15% FBS(Thermo Scientific Hyclone; Logan, UT), 2mM L-glutamine, 0.1mM non-essential amino acids, 1mM sodium pyruvate, 0.1mM B-mercaptoethanol, 100 U/ml penicillin, 100 mg/ml streptomycin, and 1000 units/ml LIF. Cells were differentiated for 5 days as embryoid bodies (EBs) formed in hanging drops of ES cell medium without LIF. Five day EBs were dissociated into single cells with 0.25% trypsin for 5 min at 37°C(13).
Cell differentiation was confirmed by a decrease over time in the proportion of cells positive for SSEA-1 (R & D; Minneapolis, MN) using fluorescence activated cell sorting (FACS). Cells were acquired on a FACS Aria cell sorter (BD Biosciences; San Jose, CA), and results were analyzed using DIVA software (BD Bioscience; San Jose, CA). Expression of endothelial cell and smooth muscle cell markers was detected by quantitative real time PCR (qRT-PCR). RNA extraction from the explanted scaffold was performed with an RNeasy Mini Kit (QIAGEN; Valencia, CA) according to the manufacturer’s protocol. Predesigned and validated gene-specific TaqMan Gene Expression Assays from Applied Biosystem (Foster City, CA, USA) were used in duplicate for qRT-PCR according to the manufacturer’s protocol. The genes of interest for analysis were calponin, PECAM, VEGF, and E-cadherin. qRT-PCR was performed in 96-well reaction plates using the iCycler iQ Real-Time PCR Detection System (Bio-Rad; Hercules, CA). Each qRT-PCR reaction consisted of the following steps: 2 min UNG incubation at 50°C to remove possible amplicon contamination, followed by 10 min at 95°C to activate the polymerase, and 40 cycles of 15 sec denaturing at 95°C and 1 min at 60°C of extension and annealing. Data were collected at the end of each elongation step and analyzed with iCycler iQ Real Time Detection System Software (Bio-Rad; Hercules, CA). HPRT was used as an endogenous control.
The differentiated iPS cell sheet was made using temperature-responsive dishes (Cellseed, Tokyo, Japan) and then seeded onto a biodegradable scaffold composed of PGA-P(CL/LA) with a diameter of 0.8mm.
Scaffolds were constructed from a nonwoven polyglycolic acid (PGA) mesh (Concordia Fibers; Coventry, RI) and a co-polymer sealant solution of poly-L-lactide and –ε-caprolactone (P(CL/LA)) using the dual cylinder chamber molding system as previously described (9).
TEVG implantations were performed using microsurgical technique. The scaffolds were inserted into the infrarenal IVCs of 3–4 month old, female SCID/bg mice (Jackson Laboratories; Bar Harbor, ME) as previously described (9). A total of 15 animals were implanted with TEVG. All animal experiments were done in accordance with the institutional guidelines for the use and care of animals, and the institutional review board approved the experimental procedures described.
Serial ultrasonography (Vevo® Visualsonics 770) was utilized for graft surveillance in both the seeded and unseeded groups. Prior to ultrasonography, mice were anesthetized with 1.5% inhaled isoflurane.
The grafts were analyzed at 1, 4, and 10 weeks with histology and immunohistochemistry.
Explanted grafts were pressure fixed in 10% formalin overnight and then embedded in paraffin or glycolmethacrylate using previously published methods(9). Sections were stained with hematoxylin and eosin (H&E).
Primary antibodies included mouse-anti-human SMA (Dako; Carpinteria, CA) and rabbit-anti-human vWF (Dako; Carpinteria, CA) (both of which have crossreactivity with mouse). Antibody binding was detected using a goat-anti-rabbit IgG-Alexa Fluor 488 or a goat-anti-mouse IgG-Alexa Fluor 488 (Invitrogen; Carlsbad, CA).
Explanted tissue grafts were frozen in OCT and each sectioned into forty 10µm sections using Cryocut 1800 (Leica; Buffalo Grove, IL). Excess OCT was removed by centrifugation in water. Following tissue digestion, DNA was isolated from samples using DNeasy Blood and Tissue Kit (QIAGEN; Valencia, CA) following manufacturer’s instructions. Validated gene-specific TaqMan Gene Expression Assays from Applied Biosystem (Foster City, CA, USA) predesigned to amplify the SRY gene were used in duplicate for qRT- PCR according to the manufacturer’s protocol. DNA amplification and quantification was performed using iCycler iQ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). Each qRT-PCR reaction consisted of the following steps: 2 min UNG incubation at 50°C to remove possible amplicon contamination, followed by 10 min at 95°C to activate the polymerase, and 40 cycles of 15 sec denaturing at 95°C and 1 min at 60°C of extension and annealing. Data were collected at the end of each elongation step and analyzed with iCycler iQ Real Time Detection System Software (Bio-Rad; Hercules, CA). Percentage of Y chromosome positive cells remaining on graft were then determined using quantification of SRY DNA content as previously described(14), taking advantage of the male iPS cell-seeded TEVGs having been implanted into female mice.
Y-Chromosome Fluorescent in situ Hybridization (FISH) / TUNEL Staining: FISH was performed on paraffin sections using digoxigenin-labeled mouse Y-chromosome probe detected with a Rhodamine-conjugated antibody to digoxigenin (Roche Diagnostics, Mannheim, Germany) as previously described(15, 16). TUNEL staining was performed using the DeadEnd Fluorometric TUNEL System kit following manufacturer’s instructions (Promega; Madison, WI). Combined FISH/TUNEL was performed by completing TUNEL protocol starting with equilibration buffer step following anti-digoxigenin hybridization. Image acquisition was performed using a Leica SP5 confocal microscope (Leica Microsystems; Wetzlar, Germany).
Statistical differences were measured using Student’s t test or ANOVA. P<0.05 was considered statistically significant.
Differentiation of iPS cells in vitro was confirmed using cell surface markers. FACS analysis of iPS cells demonstrated that the ratio of undifferentiated SSEA-1positive cells decreased over time after switching to differentiation medium (day 0: 81.0%, day 10: 28.7%, day 20: 4.3%) (Fig 1a). Concurrently, these differentiated cells expressed endothelial cell markers including VEGF, PECAM and E-cadherin, and the smooth muscle cell marker calponin (Fig 1b).
Next, in order to identify proper conditions for cell attachment to the scaffold, five million differentiated iPS cells were seeded onto the graft by manual pipetting. Attached cell number and seeding efficiency were then measured over time. There was no significant difference in attached cell number and seeding efficiency at 1, 3, 7 days (seeding efficiency: 1 day: 6.5±0.8%, 3 days: 7.6±2.5%, 7 days: 8.6±2.5%) (Fig 2). Because seeding efficiency was very low regardless of the incubation period, we developed a new seeding method using a sheet of differentiated iPS cells wrapped around the scaffold in order to seed cells more effectively. This new method using an iPS cell sheet significantly improved seeding efficiency compared with traditional pipette seeding (sheet: 86.5±11.8%, traditional: 4.9±1.5%) (Fig3).
After the implantation of this differentiated iPS cell sheet-seeded scaffold, all mice survived without thrombosis, aneurysm formation, graft rupture or calcification. Ultrasonography showed patent grafts at 10 weeks (Fig 4e). Histological evaluation of the grafts demonstrated endothelialization with VWF and an inner layer with SMA positive cells at 10 weeks (Fig 4c, d). As the male iPS cell-seeded graft was implanted into female mice, seeded cells were identified using Y-chromosome FISH. Some seeded cells remained on the outer layer of the graft at 1 week after implantation. At 4 weeks very small numbers of seeded cells were found. The number of seeded differentiated iPS cells quantified by real time PCR was found to decrease dramatically over time (1 wk: 42.2%, 4 wks: 10.4%, 10 wks: 9.8%) (Fig 5). A fraction of the iPS cells were found to be TUNEL positive at 1 week (Fig 6). No iPS cells were found to be co-localized with VWF or SMA positive cells at 10 weeks. We also found teratoma formation in 1 of 5 mice at 10 weeks.
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, 20). 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 (21–24). 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, 25).
Our previous studies have demonstrated the feasibility of creating TEVGs by seeding bone marrow mononuclear cells onto biodegradable tubular scaffolds (2, 3). 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, 27, 28). 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, 30). 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 (32–34). 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, 38), Parkinson’s disease (39, 40) and muscular dystrophy (41, 42). 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, 46). 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, 48).
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.
The authors would like to acknowledge funding from NIH BRP #R01 HL069368 (to C.K.B. and T. S.), American Heart Association Postdoctoral Fellowship (to N.H.), and Howard Hughes Medical Institute Medical Research Training Fellowship (to D.R.D). The authors thank Lin Wang (lab of Diane S. Krause) for assisting with Y chromosome FISH analysis.
The authors thank RIKEN BRC for providing iPS cells.
Presented at the 91st annual meeting of The American Association for Thoracic Surgery, Philadelphia, PA, USA