Endovascular transplantation of mRNA-enhanced mesenchymal stromal cells results in superior therapeutic protein expression in swine heart.

Heart failure has a poor prognosis and no curative treatment exists. Clinical trials are investigating gene- and cell-based therapies to improve cardiac function. The safe and efficient delivery of these therapies to solid organs is challenging. Herein, we demonstrate the feasibility of using an endovascular intramyocardial delivery approach to safely administer mRNA drug products and perform cell transplantation procedures in swine. Using a trans-vessel wall (TW) device, we delivered chemically modified mRNAs (modRNA) and mRNA-enhanced mesenchymal stromal cells expressing vascular endothelial growth factor A (VEGF-A) directly to the heart. We monitored and mapped the cellular distribution, protein expression, and safety tolerability of such an approach. The delivery of modRNA-enhanced cells via the TW device with different flow rates and cell concentrations marginally affect cell viability and protein expression in situ. Implanted cells were found within the myocardium for at least 3 days following administration, without the use of immunomodulation and minimal impact on tissue integrity. Finally, we could increase the protein expression of VEGF-A over 500-fold in the heart using a cell-mediated modRNA delivery system compared with modRNA delivered in saline solution. Ultimately, this method paves the way for future research to pioneer new treatments for cardiac disease.

Placental growth factor exerts a dual function for cardiomyogenesis and vasculogenesis during heart development.

Cardiogenic growth factors play important roles in heart development. Placental growth factor (PLGF) has previously been reported to have angiogenic effects; however, its potential role in cardiogenesis has not yet been determined. We analyze single-cell RNA-sequencing data derived from human and primate embryonic hearts and find PLGF shows a biphasic expression pattern, as it is expressed specifically on ISL1+ second heart field progenitors at an earlier stage and on vascular smooth muscle cells (SMCs) and endothelial cells (ECs) at later stages. Using chemically modified mRNAs (modRNAs), we generate a panel of cardiogenic growth factors and test their effects on enhancing cardiomyocyte (CM) and EC induction during different stages of human embryonic stem cell (hESC) differentiations. We discover that only the application of PLGF modRNA at early time points of hESC-CM differentiation can increase both CM and EC production. Conversely, genetic deletion of PLGF reduces generation of CMs, SMCs and ECs in vitro. We also confirm in vivo beneficial effects of PLGF modRNA for development of human heart progenitor-derived cardiac muscle grafts on murine kidney capsules. Further, we identify the previously unrecognized PLGF-related transcriptional networks driven by EOMES and SOX17. These results shed light on the dual cardiomyogenic and vasculogenic effects of PLGF during heart development.

Enhanced adipose-derived stem cells with IGF-1-modified mRNA promote wound healing following corneal injury.

The cornea serves as an important barrier structure to the eyeball and is vulnerable to injuries, which may lead to scarring and blindness if not treated promptly. To explore an effective treatment that could achieve multi-dimensional repair of the injured cornea, the study herein innovatively combined modified mRNA (modRNA) technologies with adipose-derived mesenchymal stem cells (ADSCs) therapy, and applied IGF-1 modRNA (modIGF1)-engineered ADSCs (ADSCmodIGF1) to alkali-burned corneas in mice. The therapeutic results showed that ADSCmodIGF1 treatment could achieve the most extensive recovery of corneal morphology and function when compared not only with simple ADSCs but also IGF-1 protein eyedrops, which was reflected by the healing of corneal epithelium and limbus, the inhibition of corneal stromal fibrosis, angiogenesis and lymphangiogenesis, and also the repair of corneal nerves. In vitro experiments further proved that ADSCmodIGF1 could more significantly promote the activity of trigeminal ganglion cells and maintain the stemness of limbal stem cells than simple ADSCs, which were also essential for reconstructing corneal homeostasis. Through a combinatorial treatment regimen of cell-based therapy with mRNA technology, this study highlighted comprehensive repair in the damaged cornea and showed the outstanding application prospect in the treatment of corneal injury.

Engineering a conduction-consistent cardiac patch with graphene oxide modified butterfly wings and human pluripotent stem cell-derived cardiomyocytes.

Engineering a conduction-consistent cardiac patch has direct implications to biomedical research. However, there is difficulty in obtaining and maintaining a system that allows researchers to study physiologically relevant cardiac development, maturation, and drug screening due to the issues around inconsistent contractions of cardiomyocytes. Butterfly wings have special nanostructures arranged in parallel, which could help generate the alignment of cardiomyocytes to better mimic the natural heart tissue structure. Here, we construct a conduction-consistent human cardiac muscle patch by assembling human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on graphene oxide (GO) modified butterfly wings. We also show this system functions as a versatile model to study human cardiomyogenesis by assembling human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSC-CPCs) on the GO modified butterfly wings. The GO modified butterfly wing platform facilitated the parallel orientation of hiPSC-CMs, enhanced relative maturation as well as improved conduction consistency of the cardiomyocytes. In addition, GO modified butterfly wings enhanced the proliferation and maturation characteristics of the hiPSC-CPCs. In accordance with data obtained from RNA-sequencing and gene signatures, assembling hiPSC-CPCs on GO modified butterfly wings stimulated the differentiation of the progenitors into relatively mature hiPSC-CMs. These characteristics and capabilities of GO modified butterfly wings make them an ideal platform for heart research and drug screening.

Engineering a conduction-consistent cardiac patch with rGO/PLCL electrospun nanofibrous membranes and human iPSC-derived cardiomyocytes.

The healthy human heart has special directional arrangement of cardiomyocytes and a unique electrical conduction system, which is critical for the maintenance of effective contractions. The precise arrangement of cardiomyocytes (CMs) along with conduction consistency between CMs is essential for enhancing the physiological accuracy of in vitro cardiac model systems. Here, we prepared aligned electrospun rGO/PLCL membranes using electrospinning technology to mimic the natural heart structure. The physical, chemical and biocompatible properties of the membranes were rigorously tested. We next assembled human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) on electrospun rGO/PLCL membranes in order to construct a myocardial muscle patch. The conduction consistency of cardiomyocytes on the patches were carefully recorded. We found that cells cultivated on the electrospun rGO/PLCL fibers presented with an ordered and arranged structure, excellent mechanical properties, oxidation resistance and effective guidance. The addition of rGO was found to be beneficial for the maturation and synchronous electrical conductivity of hiPSC-CMs within the cardiac patch. This study verified the possibility of using conduction-consistent cardiac patches to enhance drug screening and disease modeling applications. Implementation of such a system could one day lead to in vivo cardiac repair applications.

Transient secretion of VEGF protein from transplanted hiPSC-CMs enhances engraftment and improves rat heart function post MI.

Cell-based therapies offer an exciting and novel treatment for heart repair following myocardial infarction (MI). However, these therapies often suffer from poor cell viability and engraftment rates, which involve many factors, including the hypoxic conditions of the infarct environment. Meanwhile, vascular endothelial growth factor (VEGF) has previously been employed as a therapeutic agent to limit myocardial damage and simultaneously induce neovascularization. This study took an approach to transiently overexpress VEGF protein, in a controlled manner, by transfecting human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with VEGF mRNA prior to transplantation. The conditioning of iPSC-CMs with VEGF mRNA ultimately led to greater survival rates of the transplanted cells, which promoted a stable vascular network in the grafted region. Furthermore, bulk RNA transcriptomics data and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) and AGE-RAGE signaling pathways were significantly upregulated in the VEGF-treated iPSC-CMs group. The over-expression of VEGF from iPSC-CMs stimulated cell proliferation and partially attenuated the hypoxic environment in the infarcted area, resulting in reduced ventricular remodeling. This study provides a valuable solution for the survival of transplanted cells in tissue-engineered heart regeneration and may further promote the application of modified mRNA (modRNA) in the field of tissue engineering.

Epicardium-derived cells organize through tight junctions to replenish cardiac muscle in salamanders.

The contribution of the epicardium, the outermost layer of the heart, to cardiac regeneration has remained controversial due to a lack of suitable analytical tools. By combining genetic marker-independent lineage-tracing strategies with transcriptional profiling and loss-of-function methods, we report here that the epicardium of the highly regenerative salamander species Pleurodeles waltl has an intrinsic capacity to differentiate into cardiomyocytes. Following cryoinjury, CLDN6+ epicardium-derived cells appear at the lesion site, organize into honeycomb-like structures connected via focal tight junctions and undergo transcriptional reprogramming that results in concomitant differentiation into de novo cardiomyocytes. Ablation of CLDN6+ differentiation intermediates as well as disruption of their tight junctions impairs cardiac regeneration. Salamanders constitute the evolutionarily closest species to mammals with an extensive ability to regenerate heart muscle and our results highlight the epicardium and tight junctions as key targets in efforts to promote cardiac regeneration.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure.

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

Yohimbine Directly Induces Cardiotoxicity on Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Yohimbine is a highly selective and potent α2-adrenoceptor antagonist, which is usually treated as an adjunction for impotence, as well for weight loss and natural bodybuilding aids. However, it was recently reported that Yohimbine causes myocardial injury and controversial results were reported in the setting of cardiac diseases. Here, we used human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to explore electrophysiologic characterization after exposure to Yohimbine. HiPSC-CMs were differentiated by employment of inhibitory Wnt compounds. For analysis of electrophysiological properties, conventional whole-cell patch-clamp recording was used. Specifically, spontaneous action potentials, pacemaker currents (If), sodium (Na+) channel (INa), and calcium (Ca++) channel currents (ICa) were assessed in hiPSC-CMs after exposure to Yohimbine. HiPSC-CMs expressed sarcomeric-α-actinin and MLC2V proteins, as well as exhibited ventricular-like spontaneous action potential waveform. Yohimbine inhibited frequency of hiPSC-CMs spontaneous action potentials and significantly prolonged action potential duration in a dose-dependent manner. In addition, rest potential, threshold potential, amplitude, and maximal diastolic potential were decreased, whereas APD50/APD90 was prolonged. Yohimbine inhibited the amplitude of INa in low doses (IC50 = 14.2 µM, n = 5) and inhibited ICa in high doses (IC50 = 139.7 µM, n = 5). Whereas Yohimbine did not affect the activation curves, treatment resulted in left shifts in inactivation curves of both Na+ and Ca++ channels. Here, we show that Yohimbine induces direct cardiotoxic effects on spontaneous action potentials of INa and ICa in hiPSC-CMs. Importantly, these effects were not mediated by α2-adrenoceptor signaling. Our results strongly suggest that Yohimbine directly and negatively affects electrophysiological properties of human cardiomyocytes. These findings are highly relevant for potential application of Yohimbine in patients with atrioventricular conduction disorder.

An mRNA assay system demonstrates proteasomal-specific degradation contributes to cardiomyopathic phospholamban null mutation.

BACKGROUND: The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics. METHODS: A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing. RESULTS: Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes. CONCLUSION: Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.

Amnion signals are essential for mesoderm formation in primates.

Embryonic development is largely conserved among mammals. However, certain genes show divergent functions. By generating a transcriptional atlas containing >30,000 cells from post-implantation non-human primate embryos, we uncover that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 results in non-human primate embryos which do not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos display a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirm a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development.

Dexmedetomidine exhibits antiarrhythmic effects on human-induced pluripotent stem cell-derived cardiomyocytes through a Na/Ca channel-mediated mechanism.

BACKGROUND: Ventricular-like human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibit the electrophysiological characteristics of spontaneous beating. Previous studies demonstrated that dexmedetomidine (DMED), a highly selective and widely used α2-adrenoceptor agonist for sedation, analgesia, and stress management, may induce antiarrhythmic effects, especially ventricular tachycardia. However, the underlying mechanisms of the DMED-mediated antiarrhythmic effects remain to be fully elucidated. METHODS: A conventional patch-clamp recording method was used to investigate the direct effects of DMED on spontaneous action potentials, pacemaker currents (I f), potassium (K+) channel currents (I K1 and I Kr), sodium (Na+) channel currents (I Na), and calcium (Ca2+) channel currents (I Ca) in ventricular-like hiPSC-CMs. RESULTS: DMED dose-dependently altered the frequency of ventricular-like spontaneous action potentials with a half-maximal inhibitory concentration (IC50) of 27.9 µM (n=6) and significantly prolonged the action potential duration at 90% repolarization (APD90). DMED also inhibited the amplitudes of the I Na and I Ca without affecting the activation and inactivation curves of these channels. DMED decreased the time constant of the Na+ and Ca2+ channel activation at potential -40 to -20 mv, and -20 mv. DMED increased the time constant of inactivation of the Na+ and Ca2+ channels. However, DMED did not affect the I K1, I Kr, I f, and their current-voltage relationship. The ability of DMED to decrease the spontaneous action potential frequency and the Na+ and Ca2+ channel amplitudes, were not blocked by yohimbine, idazoxan, or phentolamine. CONCLUSIONS: DMED could inhibit the frequency of spontaneous action potentials and decrease the I Na and I Ca of hiPSC-CMs via mechanisms that were independent of the α2-adrenoceptor, the imidazoline receptor, and the α1-adrenoceptor. These inhibitory effects on hiPSC-CMs may contribute to the antiarrhythmic effects of DMED.

BMP-2 and VEGF-A modRNAs in collagen scaffold synergistically drive bone repair through osteogenic and angiogenic pathways.

Bone has a remarkable potential for self-healing and repair, yet several injury types are non-healing even after surgical or non-surgical treatment. Regenerative therapies that induce bone repair or improve the rate of recovery are being intensely investigated. Here, we probed the potential of bone marrow stem cells (BMSCs) engineered with chemically modified mRNAs (modRNA) encoding the hBMP-2 and VEGF-A gene to therapeutically heal bone. Induction of osteogenesis from modRNA-treated BMSCs was confirmed by expression profiles of osteogenic related markers and the presence of mineralization deposits. To test for therapeutic efficacy, a collagen scaffold inoculated with modRNA-treated BMSCs was explored in an in vivo skull defect model. We show that hBMP-2 and VEGF-A modRNAs synergistically drive osteogenic and angiogenic programs resulting in superior healing properties. This study exploits chemically modified mRNAs, together with biomaterials, as a potential approach for the clinical treatment of bone injury and defects.

Human adipose-derived stem cells enriched with VEGF-modified mRNA promote angiogenesis and long-term graft survival in a fat graft transplantation model.

BACKGROUND: Fat grafting, as a standard treatment for numerous soft tissue defects, remains unpredictable and technique-dependent. Human adipose-derived stem cells (hADSCs) are promising candidates for cell-assisted therapy to improve graft survival. As free-living fat requires nutritional and respiratory sources to thrive, insufficient and unstable vascularization still impedes hADSC-assisted therapy. Recently, cytotherapy combined with modified mRNA (modRNA) encoding vascular endothelial growth factor (VEGF) has been applied for the treatment of ischemia-related diseases. Herein, we hypothesized that VEGF modRNA (modVEGF)-engineered hADSCs could robustly enhance fat survival in a fat graft transplantation model. METHODS: hADSCs were acquired from lipoaspiration and transfected with modRNAs. Transfection efficiency and expression kinetics of modRNAs in hADSCs were first evaluated in vitro. Next, we applied an in vivo Matrigel plug assay to assess the viability and angiogenic potential of modVEGF-engineered hADSCs at 1 week post-implantation. Finally, modVEGF-engineered hADSCs were co-transplanted with human fat in a murine model to analyze the survival rate, re-vascularization, proliferation, fibrosis, apoptosis, and necrosis of fat grafts over long-term follow-up. RESULTS: Transfections of modVEGF in hADSCs were highly tolerable as the modVEGF-engineered hADSCs facilitated burst-like protein production of VEGF in both our in vitro and in vivo models. modVEGF-engineered hADSCs induced increased levels of cellular proliferation and proangiogenesis when compared to untreated hADSCs in both ex vivo and in vivo assays. In a fat graft transplantation model, we provided evidence that modVEGF-engineered hADSCs promote the optimal potency to preserve adipocytes, especially in the long-term post-transplantation phase. Detailed histological analysis of fat grafts harvested at 15, 30, and 90 days following in vivo grafting suggested the release of VEGF protein from modVEGF-engineered hADSCs significantly improved neo-angiogenesis, vascular maturity, and cell proliferation. The modVEGF-engineered hADSCs also significantly mitigated the presence of fibrosis, apoptosis, and necrosis of grafts when compared to the control groups. Moreover, modVEGF-engineered hADSCs promoted graft survival and cell differentiation abilities, which also induced an increase in vessel formation and the number of surviving adipocytes after transplantation. CONCLUSION: This current study demonstrates the employment of modVEGF-engineered hADSCs as an advanced alternative to the clinical treatment involving soft-tissue reconstruction and rejuvenation.

Intrinsic Color Sensing System Allows for Real-Time Observable Functional Changes on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Stem-cell based in vitro differentiation for disease modeling offers great value to explore the molecular and functional underpinnings driving many types of cardiomyopathy and congenital heart diseases. Nevertheless, one major caveat in the application of in vitro differentiation of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) involves the immature phenotype of the CMs. Most of the existing methods need complex apparatus and require laborious procedures in order to monitor the cardiac differentiation/maturation process and often result in cell death. Here we developed an intrinsic color sensing system utilizing a microgroove structural color methacrylated gelatin film, which allows us to monitor the cardiac differentiation process of hiPSC-derived cardiac progenitor cells in real time. Subsequently this system can be employed as an assay system to live monitor induced functional changes on hiPSC-CMs stemming from drug treatment, the effects of which are simply revealed through color diversity. Our research shows that early intervention of cardiac differentiation through simple physical cues can enhance cardiac differentiation and maturation to some extent. Our system also simplifies the previous complex experimental processes for evaluating the physiological effects of successful differentiation and drug treatment and lays a solid foundation for future transformational applications.

Cardiac progenitors and paracrine mediators in cardiogenesis and heart regeneration.

The mammalian hearts have the least regenerative capabilities among tissues and organs. As such, heart regeneration has been and continues to be the ultimate goal in the treatment against acquired and congenital heart diseases. Uncovering such a long-awaited therapy is still extremely challenging in the current settings. On the other hand, this desperate need for effective heart regeneration has developed various forms of modern biotechnologies in recent years. These involve the transplantation of pluripotent stem cell-derived cardiac progenitors or cardiomyocytes generated in vitro and novel biochemical molecules along with tissue engineering platforms. Such newly generated technologies and approaches have been shown to effectively proliferate cardiomyocytes and promote heart repair in the diseased settings, albeit mainly preclinically. These novel tools and medicines give somehow credence to breaking down the barriers associated with re-building heart muscle. However, in order to maximize efficacy and achieve better clinical outcomes through these cell-based and/or cell-free therapies, it is crucial to understand more deeply the developmental cellular hierarchies/paths and molecular mechanisms in normal or pathological cardiogenesis. Indeed, the morphogenetic process of mammalian cardiac development is highly complex and spatiotemporally regulated by various types of cardiac progenitors and their paracrine mediators. Here we discuss the most recent knowledge and findings in cardiac progenitor cell biology and the major cardiogenic paracrine mediators in the settings of cardiogenesis, congenital heart disease, and heart regeneration.

Cell-mediated delivery of VEGF modified mRNA enhances blood vessel regeneration and ameliorates murine critical limb ischemia.

Synthetic chemically modified mRNAs (modRNA) encoding vascular endothelial growth factor (VEGF) represents an alternative to gene therapy for the treatment of ischemic cardiovascular injuries. However, novel delivery approaches of modRNA are needed to improve therapeutic efficacy in the diseased setting. We hypothesized that cell-mediated modRNA delivery may enhance the in vivo expression kinetics of VEGF protein thus promoting more potent angiogenic effects. Here, we employed skin fibroblasts as a "proof of concept" to probe the therapeutic potential of a cell-mediated mRNA delivery system in a murine model of critical limb ischemia (CLI). We show that fibroblasts pre-treated with VEGF modRNA have the potential to fully salvage ischemic limbs. Using detailed molecular analysis we reveal that a fibroblast-VEGF modRNA combinatorial treatment significantly reduced tissue necrosis and dramatically improved vascular densities in CLI-injured limbs when compared to control and vehicle groups. Furthermore, fibroblast-delivered VEGF modRNA treatment increased the presence of Pax7+ satellite cells, indicating a possible correlation between VEGF and satellite cell activity. Our study is the first to demonstrate that a cell-mediated modRNA therapy could be an alternative advanced strategy for cardiovascular diseases.

Tissue-engineered trachea from a 3D-printed scaffold enhances whole-segment tracheal repair in a goat model.

Traditional treatment therapies for tracheal stenosis often cause severe post-operative complications. To solve the current difficulties, novel and more suitable long-term treatments are needed. A whole-segment tissue-engineered trachea (TET) representing the native goat trachea was 3D printed using a poly(caprolactone) (PCL) scaffold engineered with autologous auricular cartilage cells. The TET underwent mechanical analysis followed by in vivo implantations in order to evaluate the clinical feasibility and potential. The 3D-printed scaffolds were successfully cellularized, as observed by scanning electron microscopy. Mechanical force compression studies revealed that both PCL scaffolds and TETs have a more robust compressive strength than does the native trachea. In vivo implantation of TETs in the experimental group resulted in significantly higher mean post-operative survival times, 65.00 ± 24.01 days (n = 5), when compared with the control group, which received autologous trachea grafts, 17.60 ± 3.51 days (n = 5). Although tracheal narrowing was confirmed by bronchoscopy and computed tomography examination in the experimental group, tissue necrosis was only observed in the control group. Furthermore, an encouraging epithelial-like tissue formation was observed in the TETs after transplantation. This large animal study provides potential preclinical evidence around the employment of an orthotopic transplantation of a whole 3D-printed TET.