Harnessing the potential of monocytes/macrophages to regenerate tissue-engineered vascular grafts.

Cell-free tissue-engineered vascular grafts provide a promising alternative to treat cardiovascular disease, but timely endothelialization is essential for ensuring patency and proper functioning post-implantation. Recent studies from our lab showed that blood cells like monocytes (MCs) and macrophages (Mϕ) may contribute directly to cellularization and regeneration of bioengineered arteries in small and large animal models. While MCs and Mϕ are leucocytes that are part of the innate immune response, they share common developmental origins with endothelial cells (ECs) and are known to play crucial roles during vessel formation (angiogenesis) and vessel repair after inflammation/injury. They are highly plastic cells that polarize into pro-inflammatory and anti-inflammatory phenotypes upon exposure to cytokines and differentiate into other cell types, including EC-like cells, in the presence of appropriate chemical and mechanical stimuli. This review focuses on the developmental origins of MCs and ECs; the role of MCs and Mϕ in vessel repair/regeneration during inflammation/injury; and the role of chemical signalling and mechanical forces in Mϕ inflammation that mediates vascular graft regeneration. We postulate that comprehensive understanding of these mechanisms will better inform the development of strategies to coax MCs/Mϕ into endothelializing the lumen and regenerate the smooth muscle layers of cell-free bioengineered arteries and veins that are designed to treat cardiovascular diseases and perhaps the native vasculature as well.

Mitochondrial transfer mediates endothelial cell engraftment through mitophagy.

Ischaemic diseases such as critical limb ischaemia and myocardial infarction affect millions of people worldwide1. Transplanting endothelial cells (ECs) is a promising therapy in vascular medicine, but engrafting ECs typically necessitates co-transplanting perivascular supporting cells such as mesenchymal stromal cells (MSCs), which makes clinical implementation complicated2,3. The mechanisms that enable MSCs to facilitate EC engraftment remain elusive. Here we show that, under cellular stress, MSCs transfer mitochondria to ECs through tunnelling nanotubes, and that blocking this transfer impairs EC engraftment. We devised a strategy to artificially transplant mitochondria, transiently enhancing EC bioenergetics and enabling them to form functional vessels in ischaemic tissues without the support of MSCs. Notably, exogenous mitochondria did not integrate into the endogenous EC mitochondrial pool, but triggered mitophagy after internalization. Transplanted mitochondria co-localized with autophagosomes, and ablation of the PINK1-Parkin pathway negated the enhanced engraftment ability of ECs. Our findings reveal a mechanism that underlies the effects of mitochondrial transfer between mesenchymal and endothelial cells, and offer potential for a new approach for vascular cell therapy.

Near infra-red labelling and tracking of corneal endothelial cells in-vivo.

Following corneal transplantation, there is an initial, rapid decline in corneal endothelial cells (CECs) following surgery. Direct imaging of post-transplantation endothelial cells is only possible weeks after surgery and with a limited field of view. We have developed a labelling approach using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DIR) dye solution, that enables tracking of labelled CECs in vivo for at least 1 month. Initial in vitro optimization, with assessments of dye concentration on fluorescence, cellular toxicity and cell migration, performed in propagated primary CECs. Subsequently, in vivo evaluation of cellular labelling was assessed within a rabbit wound healing model. Finally, real-time visualization of human cadaver donor tissue incubated in DIR transplanted into rabbits was achieved using a clinical confocal microscope. Results revealed detectable fluorescence increased with concentration to a plateau of 100 µg/ml, with no toxicity of CECs at any concentration evaluated. DIR-labelled CECs were detectable in vivo up to 1 month, and transplanted labelled donor graft could be visualized and were trackable in vivo. Acute endothelial rejection in 1 rabbit was evidenced by detectable DIR positive cells within the anterior chamber. DIR imaging allowed for detailed imaging of the transplanted human corneal endothelium, and enabled non-invasive observation of the corneal endothelial morphology following transplantation.

Approaches for corneal endothelium regenerative medicine.

The state of the art therapy for treating corneal endothelial disease is transplantation. Advances in the reproducibility and accessibility of surgical techniques are increasing the number of corneal transplants, thereby causing a global deficit of donor corneas and leaving 12.7 million patients with addressable visual impairment. Approaches to regenerate the corneal endothelium offer a solution to the current tissue scarcity and a treatment to those in need. Methods for generating corneal endothelial cells into numbers that could address the current tissue shortage and the possible strategies used to deliver them have now become a therapeutic reality with clinical trials taking place in Japan, Singapore and Mexico. Nevertheless, there is still a long way before such therapies are approved by regulatory bodies and become clinical practice. Moreover, acellular corneal endothelial graft equivalents and certain drugs could provide a treatment option for specific disease conditions without the need of donor tissue or cells. Finally, with the emergence of gene modulation therapies to treat corneal endothelial disease, it would be possible to treat presymptomatic patients or those presenting early symptoms, drastically reducing the need for donor tissue. It is necessary to understand the most recent developments in this rapidly evolving field to know which conditions could be treated with which approach. This article provides an overview of the current and developing regenerative medicine therapies to treat corneal endothelial disease and provides the necessary guidance and understanding towards the treatment of corneal endothelial disease.

Restoring anatomical complexity of a left ventricle wall as a step toward bioengineering a human heart with human induced pluripotent stem cell-derived cardiac cells.

The heart is a highly complex, multicellular solid organ with energy-demanding processes that require a dense vascular network, extensive cell-cell interactions, and extracellular matrix (ECM)-mediated crosstalk among heterogeneous cell populations. Here, we describe the regeneration of left ventricular (LV) wall using decellularized whole rabbit heart scaffolds recellularized exclusively with human induced pluripotent stem cell-derived endothelial cells, cardiomyocytes, and other cardiac cell types. Cells were sequentially delivered to the scaffold using an optimized endothelial cell:cardiomyocyte media. Macroscopic assessment after 60 days showed that the LV wall of recellularized hearts was anatomically restored to full thickness from base to apex and endocardium to epicardium. Histologic analysis of the recellularized LV wall revealed a heterogeneous pool of cardiac cells containing aligned cardiac troponin T-positive cells in close contact with ECM; vessels varied from large artery-like, surrounded by smooth muscle actin+ cells, to capillary-like. Vessel patency was demonstrated after perfusion of recellularized hearts transplanted into the femoral artery bed of a pig. The construct exhibited visible beating and responded to chronotropic drug administration. These results demonstrate the ability to tissue engineer a vascularized, full-thickness LV wall with an unparalleled level of microanatomical organization and multicellular composition, using decellularized ECM and human cardiomyocytes, endothelial cells, and other cardiac cell types. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrix (ECM) is a bioactive template for tissue engineering, but recellularizing acellular whole heart scaffolds is challenging. Here, we successfully revascularized and repopulated a large, full-thickness portion of a ventricle using human induced pluripotent stem cell-derived endothelial and cardiac cells. At 60 days, histologic studies showed that the microanatomical organization and cellular composition of this region was similar to that of the native heart. The recellularized heart showed visible beating and responded appropriately to heartbeat-altering drugs. Vessels surrounded by smooth muscle cells and endothelial cells supported blood flow through the vessels of a recellularized heart that was surgically connected to a pig femoral artery. These findings move this approach closer to the possibility of clinical translation.

Ex vivo expansion and characterization of human corneal endothelium for transplantation: a review.

The corneal endothelium plays a key role in maintaining corneal transparency. Its dysfunction is currently treated with penetrating or lamellar keratoplasty. Advanced cell therapy methods seek to address the persistent global deficiency of donor corneas by enabling the renewal of the endothelial monolayer with tissue-engineered grafts. This review provides an overview of recently published literature on the preparation of endothelial grafts for transplantation derived from cadaveric corneas that have developed over the last decade (2010-2021). Factors such as the most suitable donor parameters, culture substrates and media, endothelial graft storage conditions, and transplantation methods are discussed. Despite efforts to utilize alternative cellular sources, such as induced pluripotent cells, cadaveric corneas appear to be the best source of cells for graft preparation to date. However, native endothelial cells have a limited natural proliferative capacity, and they often undergo rapid phenotype changes in ex vivo culture. This is the main reason why no culture protocol for a clinical-grade endothelial graft prepared from cadaveric corneas has been standardized so far. Currently, the most established ex vivo culture protocol involves the peel-and-digest method of cell isolation and cell culture by the dual media method, including the repeated alternation of high and low mitogenic conditions. Culture media are enriched by additional substances, such as signaling pathway (Rho-associated protein kinase, TGF-ß, etc.) inhibitors, to stimulate proliferation and inhibit unwanted morphological changes, particularly the endothelial-to-mesenchymal transition. To date, this promising approach has led to the development of endothelial grafts for the first in-human clinical trial in Japan. In addition to the lack of a standard culture protocol, endothelial-specific markers are still missing to confirm the endothelial phenotype in a graft ready for clinical use. Because the corneal endothelium appears to comprise phenotypically heterogeneous populations of cells, the genomic and proteomic expression of recently proposed endothelial-specific markers, such as Cadherin-2, CD166, or SLC4A11, must be confirmed by additional studies. The preparation of endothelial grafts is still challenging today, but advances in tissue engineering and surgery over the past decade hold promise for the successful treatment of endothelial dysfunctions in more patients worldwide.

Regeneration of infarcted mouse hearts by cardiovascular tissue formed via the direct reprogramming of mouse fibroblasts.

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.

Induced endothelial cells from peripheral arterial disease patients and neonatal fibroblasts have comparable angiogenic properties.

Induced endothelial cells (iECs) generated from neonatal fibroblasts via transdifferentiation have been shown to have pro-angiogenic properties and are a potential therapy for peripheral arterial disease (PAD). It is unknown if iECs can be generated from fibroblasts collected from PAD patients and whether these cells are pro-angiogenic. In this study fibroblasts were collected from four PAD patients undergoing carotid endarterectomies. These cells, and neonatal fibroblasts, were transdifferentiated into iECs using modified mRNA. Endothelial phenotype and pro-angiogenic cytokine secretion were investigated. NOD-SCID mice underwent surgery to induce hindlimb ischaemia in a murine model of PAD. Mice received intramuscular injections with either control vehicle, or 1 × 106 neonatal-derived or 1 × 106 patient-derived iECs. Recovery in perfusion to the affected limb was measured using laser Doppler scanning. Perfusion recovery was enhanced in mice treated with neonatal-derived iECs and in two of the three patient-derived iEC lines investigated in vivo. Patient-derived iECs can be successfully generated from PAD patients and for specific patients display comparable pro-angiogenic properties to neonatal-derived iECs.

Reversal of emphysema by restoration of pulmonary endothelial cells.

Chronic obstructive pulmonary disease (COPD) is marked by airway inflammation and airspace enlargement (emphysema) leading to airflow obstruction and eventual respiratory failure. Microvasculature dysfunction is associated with COPD/emphysema. However, it is not known if abnormal endothelium drives COPD/emphysema pathology and/or if correcting endothelial dysfunction has therapeutic potential. Here, we show the centrality of endothelial cells to the pathogenesis of COPD/emphysema in human tissue and using an elastase-induced murine model of emphysema. Airspace disease showed significant endothelial cell loss, and transcriptional profiling suggested an apoptotic, angiogenic, and inflammatory state. This alveolar destruction was rescued by intravenous delivery of healthy lung endothelial cells. Leucine-rich α-2-glycoprotein-1 (LRG1) was a driver of emphysema, and deletion of Lrg1 from endothelial cells rescued vascular rarefaction and alveolar regression. Hence, targeting endothelial cell biology through regenerative methods and/or inhibition of the LRG1 pathway may represent strategies of immense potential for the treatment of COPD/emphysema.

Hypoimmune induced pluripotent stem cell-derived cell therapeutics treat cardiovascular and pulmonary diseases in immunocompetent allogeneic mice.

The emerging field of regenerative cell therapy is still limited by the few cell types that can reliably be differentiated from pluripotent stem cells and by the immune hurdle of commercially scalable allogeneic cell therapeutics. Here, we show that gene-edited, immune-evasive cell grafts can survive and successfully treat diseases in immunocompetent, fully allogeneic recipients. Transplanted endothelial cells improved perfusion and increased the likelihood of limb preservation in mice with critical limb ischemia. Endothelial cell grafts transduced to express a transgene for alpha1-antitrypsin (A1AT) successfully restored physiologic A1AT serum levels in mice with genetic A1AT deficiency. This cell therapy prevented both structural and functional changes of emphysematous lung disease. A mixture of endothelial cells and cardiomyocytes was injected into infarcted mouse hearts, and both cell types orthotopically engrafted in the ischemic areas. Cell therapy led to an improvement in invasive hemodynamic heart failure parameters. Our study supports the development of hypoimmune, universal regenerative cell therapeutics for cost-effective treatments of major diseases.

Mixing Cells for Vascularized Kidney Regeneration.

The worldwide rise in prevalence of chronic kidney disease (CKD) demands innovative bio-medical solutions for millions of kidney patients. Kidney regenerative medicine aims to replenish tissue which is lost due to a common pathological pathway of fibrosis/inflammation and rejuvenate remaining tissue to maintain sufficient kidney function. To this end, cellular therapy strategies devised so far utilize kidney tissue-forming cells (KTFCs) from various cell sources, fetal, adult, and pluripotent stem-cells (PSCs). However, to increase engraftment and potency of the transplanted cells in a harsh hypoxic diseased environment, it is of importance to co-transplant KTFCs with vessel forming cells (VFCs). VFCs, consisting of endothelial cells (ECs) and mesenchymal stem-cells (MSCs), synergize to generate stable blood vessels, facilitating the vascularization of self-organizing KTFCs into renovascular units. In this paper, we review the different sources of KTFCs and VFCs which can be mixed, and report recent advances made in the field of kidney regeneration with emphasis on generation of vascularized kidney tissue by cell transplantation.

Inverse and reciprocal regulation of p53/p21 and Bmi-1 modulates vasculogenic differentiation of dental pulp stem cells.

Dental pulp stem cells (DPSC) are capable of differentiating into vascular endothelial cells. Although the capacity of vascular endothelial growth factor (VEGF) to induce endothelial differentiation of stem cells is well established, mechanisms that maintain stemness and prevent vasculogenic differentiation remain unclear. Here, we tested the hypothesis that p53 signaling through p21 and Bmi-1 maintains stemness and inhibits vasculogenic differentiation. To address this hypothesis, we used primary human DPSC from permanent teeth and Stem cells from Human Exfoliated Deciduous (SHED) teeth as models of postnatal mesenchymal stem cells. DPSC seeded in biodegradable scaffolds and transplanted into immunodeficient mice generated mature human blood vessels invested with smooth muscle actin-positive mural cells. Knockdown of p53 was sufficient to induce vasculogenic differentiation of DPSC (without vasculogenic differentiation medium containing VEGF), as shown by increased expression of endothelial markers (VEGFR2, Tie-2, CD31, VE-cadherin), increased capillary sprouting in vitro; and increased DPSC-derived blood vessel density in vivo. Conversely, induction of p53 expression with small molecule inhibitors of the p53-MDM2 binding (MI-773, APG-115) was sufficient to inhibit VEGF-induced vasculogenic differentiation. Considering that p21 is a major downstream effector of p53, we knocked down p21 in DPSC and observed an increase in capillary sprouting that mimicked results observed when p53 was knocked down. Stabilization of ubiquitin activity was sufficient to induce p53 and p21 expression and reduce capillary sprouting. Interestingly, we observed an inverse and reciprocal correlation between p53/p21 and the expression of Bmi-1, a major regulator of stem cell self-renewal. Further, direct inhibition of Bmi-1 with PTC-209 resulted in blockade of capillary-like sprout formation. Collectively, these data demonstrate that p53/p21 functions through Bmi-1 to prevent the vasculogenic differentiation of DPSC.

microRNA-129 overexpression in endothelial cell-derived extracellular vesicle influences inflammatory response caused by myocardial ischemia/reperfusion injury.

Extracellular vesicles (EVs) have the potency to function as modulators in the process of myocardial ischemia/reperfusion (I/R) injury. This investigation was performed to decipher the mechanism of human umbilical vascular endothelial cells (HUVECs)-derived EVs in myocardial I/R injury with the involvement of microRNA-129 (miR-129). HUVECs-secreted EVs were collected and identified. An I/R mouse model was developed, and cardiomyocytes were used for vitro oxygen-glucose deprivation/reperfusion model establishment. Differentially expressed miRNAs in myocardial tissues after EV treatment were assessed using microarray analysis. The target relationship between miR-129 and toll-like receptor 4 (TLR4) was identified using a dual-luciferase assay. Gain- and loss-function studies regarding miR-129 were implemented to figure out its roles in myocardial I/R injury. Meanwhile, the activation of the nuclear factor-kappa-binding (NF-κB) p65 signaling and NOD-like receptor 3 (NLRP3) inflammasome was evaluated. EVs diminished the apoptosis of cardiomyocytes and the secretion of inflammatory factors, and all these trends were reversed by miR-129 reduction. miR-129 bound to the 3'-untranslated region of TLR4 directly. The NF-κB p65 signaling and NLRP3 inflammasome were abnormally activated after I/R injury, whose impairment after EVs was partially restored by miR-129 downregulation. This study illustrated that EVs could carry miR-129 to mitigate myocardial I/R injury via downregulating TLR4 and disrupting the NF-κB signaling and NLRP3 inflammasome.

Linalool inhibits the angiogenic activity of endothelial cells by downregulating intracellular ATP levels and activating TRPM8.

Angiogenesis crucially contributes to various diseases, such as cancer and diabetic retinopathy. Hence, anti-angiogenic therapy is considered as a powerful strategy against these diseases. Previous studies reported that the acyclic monoterpene linalool exhibits anticancer, anti-inflammatory and anti-oxidative activity. However, the effects of linalool on angiogenesis still remain elusive. Therefore, we investigated the action of (3R)-(-)-linalool, a main enantiomer of linalool, on the angiogenic activity of human dermal microvascular endothelial cells (HDMECs) by a panel of angiogenesis assays. Non-cytotoxic doses of linalool significantly inhibited HDMEC proliferation, migration, tube formation and spheroid sprouting. Linalool also suppressed the vascular sprouting from rat aortic rings. In addition, Matrigel plugs containing linalool exhibited a significantly reduced microvessel density 7 days after implantation into BALB/c mice. Mechanistic analyses revealed that linalool promotes the phosphorylation of extracellular signal-regulated kinase (ERK), downregulates the intracellular level of adenosine triphosphate (ATP) and activates the transient receptor potential cation channel subfamily M (melastatin) member (TRPM)8 in HDMECs. Inhibition of ERK signaling, supplementation of ATP and blockade of TRPM8 significantly counteracted linalool-suppressed HDMEC spheroid sprouting. Moreover, ATP supplementation completely reversed linalool-induced ERK phosphorylation. In addition, linalool-induced ERK phosphorylation inhibited the expression of bone morphogenetic protein (BMP)-2 and linalool-induced TRPM8 activation caused the inhibition of ß1 integrin/focal adhesion kinase (FAK) signaling. These findings indicate an anti-angiogenic effect of linalool, which is mediated by downregulating intracellular ATP levels and activating TRPM8.

Guide Cells Support Muscle Regeneration and Affect Neuro-Muscular Junction Organization.

Muscular regeneration is a complex biological process that occurs during acute injury and chronic degeneration, implicating several cell types. One of the earliest events of muscle regeneration is the inflammatory response, followed by the activation and differentiation of muscle progenitor cells. However, the process of novel neuromuscular junction formation during muscle regeneration is still largely unexplored. Here, we identify by single-cell RNA sequencing and isolate a subset of vessel-associated cells able to improve myogenic differentiation. We termed them 'guide' cells because of their remarkable ability to improve myogenesis without fusing with the newly formed fibers. In vitro, these cells showed a marked mobility and ability to contact the forming myotubes. We found that these cells are characterized by CD44 and CD34 surface markers and the expression of Ng2 and Ncam2. In addition, in a murine model of acute muscle injury and regeneration, injection of guide cells correlated with increased numbers of newly formed neuromuscular junctions. Thus, we propose that guide cells modulate de novo generation of neuromuscular junctions in regenerating myofibers. Further studies are necessary to investigate the origin of those cells and the extent to which they are required for terminal specification of regenerating myofibers.

Overlapping and distinct biological effects of IL-6 classic and trans-signaling in vascular endothelial cells.

IL-6 affects tissue protective/reparative and inflammatory properties of vascular endothelial cells (ECs). This cytokine can signal to cells through classic and trans-signaling mechanisms, which are differentiated based on the expression of IL-6 receptor (IL-6R) on the surface of target cells. The biological effects of these IL-6-signaling mechanisms are distinct and have implications for vascular pathologies. We have directly compared IL-6 classic and trans-signaling in ECs. Human ECs expressed IL-6R in culture and in situ in coronary arteries from heart transplants. Stimulation of human ECs with IL-6, to model classic signaling, triggered the activation of phosphatidylinositol 3-kinase (PI3K)-Akt and ERK1/2 signaling pathways, whereas stimulation with IL-6 + sIL-6R, to model trans-signaling, triggered activation of STAT3, PI3K-Akt, and ERK1/2 pathways. IL-6 classic signaling reduced persistent injury of ECs in an allograft model of vascular rejection and inhibited cell death induced by growth factor withdrawal. When inflammatory effects were examined, IL-6 classic signaling did not induce ICAM or CCL2 expression but was sufficient to induce secretion of CXCL8 and support transmigration of neutrophil-like cells. IL-6 trans-signaling induced all inflammatory effects studied. Our findings show that IL-6 classic and trans-signaling have overlapping but distinct properties in controlling EC survival and inflammatory activation. This has implications for understanding the effects of IL-6 receptor-blocking therapies as well as for vascular responses in inflammatory and immune conditions.

Preservation of microvascular integrity and immunomodulatory property of prevascularized human mesenchymal stem cell sheets.

Prevascularization is essential to ensure the viability, functionality, and successful integration of tissue-engineered three-dimensional (3D) constructs with surrounding host tissues after transplantation. Human mesenchymal stem cell (hMSC) sheet can be prevascularized by coculturing with endothelial cells (ECs), and then be further used as building blocks for engineering 3D complex tissues. In addition, predifferentiation of hMSCs into a tissue-specific lineage in vitro has been proven to promote graft engraftment and regeneration. However, it is unclear if the prevascularized hMSC sheets can still maintain their microvascular integrity as well as the immune-regulatory properties after their tissue-specific differentiation. The objective of this study was to investigate the effects of differentiation cues on the microvascular structure, angiogenic factor secretion, and immunogenic responses of prevascularized hMSC sheets. The results showed that upon coculturing with ECs, hMSC sheets successfully formed microvascular network, while maintaining hMSCs' multi-lineage differentiation capability. The next step, osteogenic and adipogenic induction, damaged the preformed microvascular structures and compromised the angiogenic factor secretion ability of hMSCs. Nonetheless, this effect was mitigated by adjusting the concentration of differentiation factors. The subcutaneous transplantation in an immunocompetent rat model demonstrated that the osteogenic differentiated prevascularized hMSC sheet preserved its microvascular structure and immunomodulatory properties comparable to the undifferentiated prevascularized hMSC sheets. This study suggested that a balanced and optimal differentiation condition can effectively promote the tissue-specific predifferentiation of prevascularized hMSC sheet while maintaining its immunomodulatory and tissue integration properties.

Coadministration of endothelial and smooth muscle cells derived from human induced pluripotent stem cells as a therapy for critical limb ischemia.

Critical limb ischemia is a condition in which tissue necrosis occurs due to arterial occlusion, resulting in limb amputation in severe cases. Both endothelial cells (ECs) and vascular smooth muscle cells (SMCs) are needed for the regeneration of peripheral arteries in ischemic tissues. However, it is difficult to isolate and cultivate primary EC and SMC from patients for therapeutic angiogenesis. Induced pluripotent stem cells (iPSCs) are regarded as useful stem cells due to their pluripotent differentiation potential. In this study, we explored the therapeutic efficacy of human iPSC-derived EC and iPSC-derived SMC in peripheral artery disease model. After the induction of mesodermal differentiation of iPSC, CD34+ progenitor cells were isolated by magnetic-activated cell sorting. Cultivation of the CD34+ progenitor cells in endothelial culture medium induced the expression of endothelial markers and phenotypes. Moreover, the CD34+ cells could be differentiated into SMC by cultivation in SMC culture medium. In a murine hindlimb ischemia model, cotransplantation of EC with SMC improved blood perfusion and increased the limb salvage rate in ischemic limbs compared to transplantation of either EC or SMC alone. Moreover, cotransplantation of EC and SMC stimulated angiogenesis and led to the formation of capillaries and arteries/arterioles in vivo. Conditioned medium derived from SMC stimulated the migration, proliferation, and tubulation of EC in vitro, and these effects were recapitulated by exosomes isolated from the SMC-conditioned medium. Together, these results suggest that iPSC-derived SMC enhance the therapeutic efficacy of iPSC-derived EC in peripheral artery disease via an exosome-mediated paracrine mechanism.

In vitro evaluation of surface biological properties of decellularized aorta for cardiovascular use.

The aim of this study was to determine an in vitro evaluation method that could directly predict in vivo performance of decellularized tissue for cardiovascular use. We hypothesized that key factors for in vitro evaluation would be found by in vitro assessment of decellularized aortas that previously showed good performance in vivo, such as high patency. We chose porcine aortas, decellularized using three different decellularization methods: sodium dodecyl-sulfate (SDS), freeze-thawing, and high-hydrostatic pressurization (HHP). Immunohistological staining, a blood clotting test, scanning electron microscopy (SEM) analysis, and recellularization of endothelial cells were used for the in vitro evaluation. There was a significant difference in the remaining extracellular matrix (ECM) components, ECM structure, and the luminal surface structure between the three decellularized aortas, respectively, resulting in differences in the recellularization of endothelial cells. On the other hand, there was no difference observed in the blood clotting test. These results suggested that the blood clotting test could be a key evaluation method for the prediction of in vivo performance. In addition, evaluation of the luminal surface structure and the recellularization experiment should be packaged as an in vitro evaluation because the long-term patency was probably affected. The evaluation approach in this study may be useful to establish regulations and a quality management system for a cardiovascular prosthesis.