The development of novel therapies that are clinically translatable is critical if one hopes to transition research from the bench to the bedside. Although our prior SMC-EPC cell sheets were potent angiogenic constructs, using the rodent model as a cell source for EPC and SMC isolation precluded translation as a realistic therapy. Our study showed that the use of human-derived EPCs and SMCs in human subjects is promising.
We introduced human-derived EPCs isolated from peripheral blood and the SMC lineage when MSCs were cultured with SMC growth medium on ECM-coated dishes. A confluent bi-level cell sheet made of human-derived EPCs and SMCs was engineered using cell sheet technology. These advantages are most probably multifactorial in nature, by which the targeted transplantation of an SMC-EPC bi-level cell sheet induced the generation of structurally mature blood vessels and increased myocardial viability in the ischemic border zone myocardium, enhanced myocardial functional recovery, and limited adverse ventricular and myocardial remodeling in the athymic rodent model of MI. The observed increase in structurally mature vessel density in the ischemic border zone myocardium elucidated the significant in vivo angiogenic potential of this technology. A significantly enhanced vasculogenic potential was observed in vitro, wherein EPCs were stimulated with SMC-conditioned culture medium, indicating biological cross-talk by co-culturing SMCs and EPCs; this effect is a mechanistic component of the augmented angiogenesis demonstrated in vivo. More importantly, the data established direct migration of the transplanted EPCs and SMCs into the host myocardium and confirmed that these cells were some elements of a newly formed vasculature using our unique cell fate tracking experiments featuring xenogeneic transplantation. Moreover, the therapeutic potential of cell sheets was reflected by the upregulation of CXCL12, Notch3, Ndnf, Adam12, and Type VI collagen in the ischemic border zone myocardium, as indicated by bulk RNA Seq. Furthermore, regarding cell engraftment, the cell-sheet-transplanted rat showed prolonged cell retention, as quantitatively assessed using USPIO-enhanced MRI. Thus, the robust angiogenic effect of bi-level cell sheets was because of the upregulation of angiogenesis-related genes; the recruitment of transplanted EPCs and SMCs improved myocardial viability, enhanced myocardial functional recovery, and induced reverse myocardial and ventricular remodeling of the ischemic heart.
Tissue engineering is a necessary tool for developing effective regenerative therapies . In the past decade, using tissue engineering techniques, various cell scaffold therapies have been studied and are commercially available currently. One widely used therapy includes scaffold-based tissue engineering, which includes the use of biodegradable scaffolds , decellularized tissues , hydrogel and cell mixtures , bioprinting ]20], 3D bio-fabrication , and fiber-based tissue engineering . Of these, our group employed the scaffold-free technology for cell-sheet engineering [23, 24].
A specialized dish that is covalently grafted with poly (N-isopropyl acrylamide) is used to create a cell sheet; poly (N-isopropyl acrylamide) is a temperature-responsive polymer which undergoes an enzyme-free hydrophobic to hydrophilic transformation by temperature lowering . Thus, using these dishes, three-dimensional tissues can be created from densely adherent cells, and an artificial scaffold or enzymatic digestion is not required. Cell sheets can be engineered easily and have an advantage of integrating within native tissue. These cell sheets preserve the cell-cell junctions and the extracellular matrix (ECM) deposited on the basal surface of the cell sheet in addition to regional morphological differences between different cell types following mobilization from the UpCell dish. SEM images showed spherical cells of the SMC layer, and a thin, film-like EPC mono-layer, consistent with the results reported previously .
Previously, using a rodent model of ischemic cardiomyopathy, we demonstrated that tissue-engineered cell sheets with rodent-derived SMCs and EPCs have advantages of the natural interactions between EPCs and SMCs, a structurally organized microvasculature, and functional recovery of distressed myocardium . However, we observed the presence of SMCs in the thoracic aorta; it was also observed that bone marrow-derived MSCs have the potential to differentiate into various cell types, including SMCs. Considering the ECM regulates SMC phenotypic modulation, fibronectin was observed to guide the differentiation of MSCs into SMCs and simultaneously preserve cellular proliferative capacity . Then, we indicated that the rodent origin bone marrow-derived, anatomically oriented bi-level cell sheet made of isolated EPCs and trans-differentiated SMCs is a multi-lineage cellular therapy, obtained from a translationally practical source. The following properties of tissue-engineered constructs make it an appropriate engineering combination for therapeutic use: maintenance of important interactions between EPCs and SMCs, thereby enhancing mature neovascularization within the border zone myocardium, minimization of post-infarction adverse remodeling, and strengthening of ventricular function in a rodent model of ischemic cardiomyopathy .
The mechanism by which SMC-EPC bi-level cell sheets induced blood vessel formation is dynamic and complex. Based on increased arterial density as shown by microscopy and preserved myocardial viability as shown by MEMRI in the ischemic border zone myocardium, the bi-level cell sheet containing human-derived EPCs and SMCs in separate layers was directly incorporated into the well-structured vasculature and remained present 8 weeks post-infarction in the cell fate tracking experiments.
This finding is consistent with our previous study . Additionally, in this study, we elucidated an alternative exogenous mechanism underlying the therapeutic effect of SMC-EPC bi-level cell sheets in the acute MI rat model. The effect is observed because of the increased expression of CXCL12, Notch3, Ndnf, Adam12, and type VI collagen, as indicated by bulk RNA Seq.
Notably, CXCL12, also known as stromal cell-derived factor 1 (SDF-1), binds selectively to two chemokine receptors, namely CXCR4 and ACKR3 [26, 27]. CXCL12 increases vasculogenesis, limits infarct size, and improves cardiac function post-MI, as we reported previously [28, 29]. Our group also reported that the CXCL12-dependent process facilitates the formation of collateral artery network and functional recovery of the heart , which supports our observations of the upregulated expression of CXCL12 and the well-structured blood vessels in the ischemic border zone myocardium after SMC-EPC bi-level cell sheet transplantation. On the other hand, stem cell migration, recruitment, and homing are regulated by the interactions among cytokines, chemokines, and extracellular matrix, and the CXCL12/CXCR4 axis plays a central role in the mobilization of stem cells and their homing to ischemic tissues [14, 15]. Thus, our findings are evidence of the direct contribution of donor’s SMCs and EPC to the neovascularization in the recipient’s ischemic myocardium.
Notch signaling is implicated in arteriogenesis  and intersects with hypoxic signaling, such as HIF1α . Our data showed that Notch3 expression was upregulated under hypoxic conditions after ischemic injury, which is explained by the aforementioned report, whereas our key finding was that Notch3 expression was further upregulated in response to cell sheet transplantation, which was verified microscopically by a traditional immunohistochemical assessment. More interestingly, we observed the presence of Notch3/α-SMA/CD31-positive blood vessel formation in the ischemic border zone myocardium 1 week after SMC-EPC bi-level cell sheet transplantation. Notch3 regulates the involvement and stability of mural cells and pericytes and affects the vascular tone in resistance arteries as structurally supporting smooth muscle cells [33, 34]. Based on these reports and our findings, possibilities include that the Notch3 signaling pathway was initiated as a maladaptive response to hypoxia post-MI and that Notch3 expression was effectively upregulated after cell sheet transplantation, indicating that Notch3 not only promotes blood vessel formation but is also implicated in the functional blood vessel by establishing vessel networks, assembling pericytes, and inducing the maturation of smooth muscle cells .
Elucidating the whole mechanism by which the SMC-EPC bi-level cell sheets limited adverse cardiac remodeling is difficult. The present study identified the reverse ventricular remodeling process, such as LV volume and LV mass, as measured using cardiac MRI images following cell sheet transplantation. In addition, cell sheet transplantation attenuated cellular hypertrophy in the non-infarcted remote myocardium, as observed microscopically. Based on these results of ventricular reverse remodeling as well as myocardial reverse remodeling, we attempted to explain the correlation between the two. First, neovascularization and the following improved myocardial viability on the targeted myocardial territory could reduce the number of necrotic/apoptotic cardiomyocytes. Next, the reduced accumulation of fibrous components would inhibit the thinning of the left ventricular wall [36, 37]. Furthermore, the preserved wall thickness could reduce wall stress of the left ventricle, theoretically leading to reduced LV volume, known as adverse cardiac remodeling . On the other hand, our data, together with the theory of Grossman et al. on volume-related cellular hypertrophy (i.e., cell thickening and elongation) , suggest that abolishing volume overload could activate cellular and extracellular mechanisms that modify myocardial structural remodeling. Moreover, as indicated by our bulk RNA sequencing, the native ECM remodeling processes were enhanced after cell sheet transplantation. Thus, we speculated that the release from volume overload could initiate a subsequent adjustable response in the molecular signals that result in the regression of myocardial hypertrophy and extracellular matrix turnover .
Cell engraftment is another critical aspect of myocardial regeneration. The present study focused on serial changes in a detailed fashion using a USPIO-enhanced MRI study on cell engraftment following cell-sheet transplantation. With regard to cell engraftment, the data demonstrated prolonged cell retention of the transplanted SMC-EPC cell sheets on the myocardium. The potential advantages of cell-sheet technology have been reported to include the delivery of a larger number of transplanted cells and the integration with native tissues without destroying the cell-cell or cell–ECM adhesions in the cell sheet [41, 42]. The degree of neovascularization in the transplanted myocardium and subsequent myocardial inflammation after cell sheet transplantation affect the retention and engraftment of transplanted cells in cell sheet therapy [43, 44]. Based on these findings and the significant findings of long-standing cell retention and the recruitment of transplanted cells from the SMC-EPC bi-level cell sheet, the SMC-EPC cell sheet might have improved the hypoxic environment in the transplanted area to a greater degree, thus potentially improving initial and long-term cell engraftment through a higher expression of associated genes .
Finally, type VI collagen not only aids cell attachment and connects with the surrounding matrix but also acts as an early sensor of the injury/repair response [46, 47], whereas type VI collagen contributes to angiogenesis . Thus, the potential mechanism is that the upregulated expression of type VI collagen in the ischemic border zone area may directly initiate neovascularization in the corresponding myocardium and contribute to maintaining cell engraftment after cell sheet transplantation.
Currently, this therapy for acute MI is not appropriate for use in clinical settings because of the time required for the isolation, cultivation, and manipulation of the cells in vitro. Despite this, this construct is a potential candidate for allogeneic therapy. The utility of these cell sheets for the treatment of chronic ischemic cardiomyopathy and diabetic cardiomyopathy is currently being studied, including the examination of the survival duration of the transplanted cells. Finally, the efficiency of bilayer cell sheets in comparison with those of EPC, SMC, or MSC monolayer cell sheets requires further studies.
In conclusion, the human-derived, anatomically oriented bi-level cell sheets made of isolated EPCs and transdifferentiated SMCs are a promising, multi-lineage cellular therapy obtained from a translationally practical source. Delivery of a tissue-engineered construct that maintains important interactions between EPCs and SMCs enhances structurally mature neovascularization within the border zone myocardium along with myocardial upregulation of the expression of angiogenesis-related genes as well as direct migration of transplanted EPCs and SMCs, increases myocardial viability, minimizes post-infarction adverse remodeling, and strengthens ventricular function. Collectively, these pathways establish transcriptome-wide changes in the left ventricular response to acute ischemia, indicating that the bi-level cell sheets gradually promote productive remodeling and prevent pathological ventricular dilation via these pathways and genes.